Antigenic protein originating in Malassezia

ABSTRACT

A substantially pure, isolated, antigenic protein from fungi of the genus  Malassezia , characterized in that said antigenic protein has a binding ability to IgE antibodies from patients with allergoses; an antigenic fragment derived from the antigenic protein; and an antibody against the antigenic protein or fragments thereof. According to the present invention, there can be provided an isolated and purified antigenic protein having high purity from  Malassezia , antigenic fragments thereof, and a specific antibody against those antigenic protein or fragments thereof. In addition, there can be provided a diagnostic agent, a therapeutic agent, or a prophylactic drug for  Malassezia  allergoses, wherein the agent includes, as an active ingredient, the antigenic protein or fragments thereof.

This application is a divisional of application Ser. No. 10/109,670filed on Apr. 1, 2002 now U.S. Pat. No. 6,911,208 which is a Divisionalof application Ser. No. 09/091,097 filed on Jun. 12, 1998, now U.S. Pat.No. 6,432,407, and for which priority is claimed under 35 U.S.C. §120.Application Ser. No. 09/091,097 is the national phase of PCTInternational Application No. PCT/JP96/03602 filed on Dec. 10, 1996under 35 U.S.C. § 371. The entire contents of each of theabove-identified applications are hereby incorporated by reference. Thisapplication also claims priority of the following: Application No.7-346627 filed in Japan on Dec. 12, 1995; Application No. 8-257612 filedin Japan on Sep. 5, 1996; and Application No. 8-257613 filed in Japan onSep. 5, 1996 under 35 U.S.C. § 119.

TECHNICAL FIELD

The present invention relates to a novel antigenic protein which isisolated and purified from Malassezia fungi, useful for diagnosis,treatment, and prophylaxis for allergoses and infectious diseases ofwhich causative microorganisms are Malassezia fungi, and to antigenicfragments thereof, an antibody against the antigenic protein orantigenic fragments thereof, and the like.

Further, the present invention relates to a recombinant Malasseziaantigenic protein, a gene encoding the antigenic protein, and also to anepitope of the protein, and the like.

BACKGROUND ART

As a result of sensitization by the causative antigen for the diseases,in many of the allergoses, an antigen (allergen)-specific IgE antibody(reagin antibody) is produced in sera and tissue. Upon re-exposure tothe same antigen, IgE bound to the mast cells or basophiles and thespecific allergen become coupled together to cause IgE crosslink on thecell surface, resulting in physiological effects due to the IgE-antigeninteraction. Such physiological effects include the release ofhistamine, serotonin, heparin, eosinophilic chemotactic factor, orvarious leukotrienes, whereby persisting constriction of bronchialsmooth muscle is caused. These released substances act as chemicalmediators to induce allergic symptoms due to a coupling of IgE and aparticular allergen. The effects of an allergen manifest themselves viathese symptoms, and such effects can occur systemically or locally,depending on the route of antigen invasion in the body and the patternof IgE sedimentation on mast cells or basophiles. Local symptomsgenerally occur on the epithelial surface at the position of allergeninvasion in the body. Systemic effects are consequences of IgE-basophileresponse to the antigen in the blood vessels, which are typicallyexemplified by anaphylactic shock. The helper T (Th) cell plays a keyrole in the series of reactions. Among the various cytokines produced byTh cells activated by antigen stimulation, IL4 promotes IgE production.

A wide variety of substances induce allergic symptoms in humans. Todate, allergens have been viewed as an assembly of a large number ofsubstances represented by pollens or house dusts. As a result of recentadvances in separation and purification techniques and methods forevaluating allergen activity, it has been clearly obvious that theallergen comprises a single substance or several kinds of principalsubstances. In particular, a rapid progress in research into allergensof Cryptomeria japonica (Japanese cedar) pollen, ticks, cats, and thelike has been made, and major allergens, such as Cry j 1 and Cry j 2have been isolated from Cryptomeria japonica pollen; Der f 1, Der f 2,and Der f 3 have been isolated from ticks; and Fel d 1 has been isolatedfrom cats. Furthermore, genes encoding these allergenic proteins havealso been isolated, thereby making it possible to prepare pureallergenic proteins in large amounts by genetic engineering techniques.

In the diagnosis of allergoses, it is necessary to first identify theantigen of which the microorganisms are causative, and in order toaccomplish this purpose, over 100 kinds of commercially availableantigen extracts, and in some cases, those prepared in-house, are firstsubjected to intracutaneous tests using suspected antigen extracts. Inthe case where an antigen of which is a very likelihood of being thecausative antigen is found, the antigen can be specifically identifiedby assaying serum IgE antibody titration by RAST method and the like,provocative tests, or histamine release tests using whole blood orlymphocytes. Because these antigen extracts do not have their potencywell titrated, however, attention should be marked to the risk ofanaphylactogenesis upon use. Usable therapies for allergoses includeantihistaminics, steroidal anti-inflammatory drugs, and mediator releasesuppressors, and the therapy of hyposensitization using a diagnosticallyspecified antigen serves excellently. It should be noted, however, thatthe currently available method of therapy of hyposensitization requiresan antigen solution to be intracutaneously administered little by littleonce or twice each week for three to four months over which period thestarting dose is escalated to a maintenance dose, which is thenmaintained for one to three years. If dose escalation is easy, it can beexpected that excellent therapeutic effects can be obtained. However,grave side reactions can occur because of the above uncertain potency ofthe antigen used, and because of the presence of various impuritysubstances therein, thereby greatly limiting its use of the antigen.

Fungi belonging to the genus Malassezia (hereinafter abbreviated as M.)are known to include M. furfur (also known as Pityrosporum ovale orPityrosporum orbiculare), M. pachydermatis, M. sympodialis, and thelike. Malassezia is reportedly commonly present on the body surfaces ofvarious animals and on those of humans. Its pathogenicity and role inallergoses have long been studied. Regarding pathogenicity, Malasseziais suspected of being causative microorganisms for dermatitis, tineaversicolor, folliculitis, dandruff, and other conditions. It is alsosuspected of being associated with allergoses, such as atopicdermatitis, and there is a great chance that it is involved in thesediseases as a causative microorganism.

Currently, antigen extracts from Malassezia are commercially available.These extracts are unpurified or partially purified products obtainedfrom cultures of M. furfur, and are thus considered complex mixturescomprising proteins, sugars, and lipids.

Conventionally, a large number of allergenic proteins from Malasseziahave been reported to be contained in such antigen extracts, including87, 76, 67, 45, 37, 28, 25, 14, 13 kDa IgE-binding proteins, which aredetected by immunoblotting using IgE antibodies in sera of patientsafter a crude extract from a Malassezia fungus is separated bySDS-polyacrylamide gel electrophoresis (PAGE) (Siv Johansson et al.,Acta Derm. Venereol., 71, 11-16, 1991; E. Jensen-Jarolim et al., J.Allergy Clin. Immunol., 89, 44-51, 1992; Zargari et al., Allergy, 49,50-56, 1994). Thus, since the proteins produced by the Malassezia fungiare beyond a wide variety of proteins, simple separation by SDS-PAGEalone is unsatisfactory, and it cannot be thought that a single proteinband in SDS-PAGE which is conventionally reported represents ahomogenous protein. In other words, because a plurality of proteinssharing the same protein band in SDS-PAGE are usually present, anIgE-binding protein, even if a single protein band is shown, must beseparated from many other proteins contained in the band, which in turnnecessitates combining with another effective separation method.Furthermore, in order to be useful for a diagnostic or therapeuticpurpose, it is necessary to isolate an antigenic protein and clarify itsantigenicity using a number of sera from patients, to identify it as themajor allergen, and to establish a method for producing it for supplyingthe desired produce with demonstrated protein chemical quality. Forthese reasons, a homogenous and single antigenic protein must beisolated by repeating separation by various chromatographies and assayof the antigen activity. The protein finally obtained needs to beconfirmed as having homogeneity in ion exchange chromatography andhomogeneity in isoelectric electrophoresis, as well as that in SDS-PAGE.

According to the above-mentioned various reports, however, suchsubstances observed in SDS-PAGE are dealt with as if they each representa single IgE-binding protein. Actually, however, no one have yet beensuccessful to isolate and purify them, and there have never beendiscussed on the identity of the band as a mixture of many mutuallyunrelated proteins. Accordingly, as a matter of course, no attempts havebeen yet made to isolate IgE-binding proteins from the complicatedmixture and confirm the antigenicity thereof as isolated proteins usingsera of patients with allergy. Further, no reports have been yet maderegarding the properties of protein chemistry or amino acid sequencesthereof. For this reason, it remains unknown as to the mutual identityor relevancy (for example, one is a decomposition product by protease ofthe other protein), and other aspects of IgE-binding proteins discussedin the above reports.

Even though the Malassezia fungi have been remarked as causativemicroorganisms for allergoses, including atopic dermatitis, as describedabove, no one have yet succeeded in isolating and purifying anIgE-binding protein from a crude extract comprising a complicatedprotein mixture. As a matter of course, the antigenicity of such anisolated protein has not been confirmed using sera of patients withallergy. Moreover, there have been no reports of the properties ofprotein chemistry or amino acid sequences thereof, and there are noreported cases on isolation of the gene encoding the above protein.

DISCLOSURE OF THE INVENTION

In order to assess the likelihood of being a causative microorganism,skin tests using crude antigens, Malassezia cell extracts as describedabove, provocative tests, quantitative assay tests for various IgEantibodies by RAST method, assay for histamine release, and the like,and other approaches are performed, in addition to microbiologicalcultivation tests. Because these crude antigens contain a large numberof different impurity substances, however, accurate diagnosis cannot bemade. In addition, when used for skin tests and provocative tests, thecrude antigen can pose a risk of development of adverse reactions, andthe like. Moreover, when using the crude antigen for therapy ofhyposensitization, there is a risk of anaphylactogenesis associatedtherewith, posing extreme limitation on the dose of the crude antigen,so that therapeutic effects cannot be expected. In addition, it is alsodifficult to use the crude antigen as a vaccine for preventinginfections. To date, there have been no successful cases on isolation ofsuch purified pure antigen from Malassezia, and there is, therefore, amajor set back on the infections caused by Malassezia fungi and thediagnosis and therapy of allergoses.

Accordingly, in consideration of the present situation, the followingobjects are achieved by the present invention.

-   (1) A first object of the present invention is to provide a    substantially pure, isolated, antigenic protein from fungi of the    genus Malassezia, namely a purified Malassezia allergen, preferably    a main allergen for patients with Malassezia allergoses, and to    provide their properties of protein chemistry. Further, the object    is also to provide a functionally equivalent antigenic protein    having properties immunologically equivalent to those of the    antigenic protein.-   (2) A second object of the present invention is to provide an    antigenic fragment having an antigenic epitope contained in these    purified antigenic proteins.-   (3) A third object of the present invention is to provide an    antibody or fragments thereof against the above antigenic protein or    antigenic fragments.-   (4) A fourth object of the present invention is to provide a    diagnostic agent for diseases, such as allergoses of which causative    microorganisms are Malassezia fungi, the diagnostic agent including,    as an active ingredient, the above antigenic protein or antigenic    fragments.-   (5) A fifth object of the present invention is to provide a    therapeutic agent for diseases, such as allergoses of which    causative microorganisms are Malassezia fungi, the therapeutic agent    including, as an active ingredient, the above antigenic protein or    antigenic fragments.-   (6) A sixth object of the present invention is to provide a method    for immunological, quantitative assay of the Malassezia allergen.-   (7) A seventh object of the present invention is to provide a novel    recombinant Malassezia antigenic protein having immunological    properties equivalent to those of the purified antigenic protein of    item (1).-   (8) A eighth object of the present invention is to provide a    polynucleotide encoding a novel recombinant Malassezia antigenic    protein.-   (9) A ninth object of the present invention is to provide an    antigenic fragment having an epitope contained in the recombinant    Malassezia antigenic protein.-   (10) A tenth object of the present invention is to provide an    antibody or fragments thereof which specifically bind to the above    recombinant Malassezia antigenic protein or antigenic fragments    thereof.-   (11) An eleventh object of the present invention is to provide a    synthesized oligonucleotide probe or a synthesized oligonucleotide    primer which hybridizes to the above polynucleotide.-   (12) A twelfth object of the present invention is to provide a    diagnostic agent for Malassezia allergoses or Malassezia infectious    diseases, including, as an active ingredient, the above recombinant    Malassezia antigenic protein or antigenic fragments thereof.-   (13) A thirteenth object of the present invention is to provide a    therapeutic agent for Malassezia allergoses or Malassezia infectious    diseases, including, as an active ingredient, the above recombinant    Malassezia antigenic protein or antigenic fragments thereof.

For the purpose of isolating Malassezia allergens useful for thediagnosis and therapy of patients with allergy with the cell componentsof M. furfur TIMM2782, a fungal strain belonging to the genusMalassezia, the present inventors have screened sera of patients withRAST-positive and positive skin tests for antigenic proteins, using cellextract crude antigens. As a result, the present inventors havesucceeded in isolating 13 kinds of antigenic proteins designated as MF-1to -13, respectively, and also succeeded in determination of the partialamino acid sequences of some of the antigenic proteins. Moreover, thepresent inventors have synthesized a polynucleotide to be used forprimers on the basis of the information for the partial amino acidsequences of the Malassezia antigenic proteins thus isolated, andcarried out polymerase chain reaction (PCR) with a cDNA derived from M.furfur cell mRNA as the starting material, using the polynucleotide as aprimer, to give a portion of the gene encoding the desired Malasseziaantigenic protein. Next, the desired gene has been isolated from an M.furfur cell cDNA library using the entire or partial fragment of thisPCR fragment as a probe. Also, an overlapping peptide has beensynthesized on the basis of the amino acid sequence of MF-1. The presentinventors have clarified that an epitope for T cell and an epitope for Bcell can be found by carrying out search for an epitope against thepatient serum IgE antibody and search for another epitope against theMF-1 monoclonal antibody, using the above peptide. The present inventionhas been completed based on the above finding.

In other words, one embodiment of the present invention relates to asubstantially pure, isolated, antigenic protein or antigenic fragmentsthereof from fungi of the genus Malassezia, characterized by having abinding ability to an IgE antibody from patients with allergoses.

Another embodiment of the present invention relates to a recombinantMalassezia antigenic protein or antigenic fragments thereof,characterized by having immunological properties functionally equivalentto those of the isolated and purified antigenic protein.

Another embodiment of the present invention relates to a polynucleotideencoding the recombinant Malassezia antigenic protein or antigenicfragments thereof of the present invention.

Another embodiment of the present invention relates to an antibody orfragments thereof against the isolated and purified antigenic protein orantigenic fragments thereof of the present invention, or against therecombinant Malassezia antigenic protein or antigenic fragments thereofof the present invention.

Another embodiment of the present invention relates to a synthesizedoligonucleotide probe or a synthesized oligonucleotide primer whichhybridizes to the polynucleotide of the present invention.

Another embodiment of the present invention relates to a diagnosticagent for Malassezia allergoses or Malassezia infectious diseases,characterized in that the diagnostic agent includes, as an activeingredient, the isolated and purified antigenic protein or antigenicfragments thereof of the present invention, or the recombinantMalassezia antigenic protein or antigenic fragments thereof of thepresent invention.

Another embodiment of the present invention relates to a therapeuticagent for Malassezia allergoses or Malassezia infectious diseases,characterized in that the therapeutic agent includes, as an activeingredient, the isolated and purified, antigenic protein or antigenicfragments thereof of the present invention, or the recombinantMalassezia antigenic protein or antigenic fragments thereof of thepresent invention.

Another embodiment of the present invention relates to a method forquantitative assay of Malassezia allergen, characterized in that theimmunological, quantitative assay of the Malassezia allergen isconducted by using the isolated and purified antigenic protein of thepresent invention, or the recombinant Malassezia antigenic protein ofthe present invention as a standard and antibodies against the aboveantigenic protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing chromatographic analysis by Mono Q of apartially purified, crude antigen 2782 of Malassezia.

FIG. 2 is a graph showing the binding ability of Mono Q fractions of apartially purified, crude antigen 2782 of Malassezia with an IgEantibody in a patient serum.

FIG. 3 is an electrophoretic analysis obtained by subjecting Mono Qfractions of a partially purified, crude antigen 2782 of Malassezia toSDS-PAGE, and then staining with CBB.

FIG. 4 is an electrophoretic analysis obtained by subjecting Mono Qfractions of a partially purified, crude antigen 2782 of Malassezia toSDS-PAGE, and then conducting immunoblotting.

FIG. 5 is a chart showing an MF-1 peak by Mono Q chromatography.

FIG. 6 is a chart showing an MF-2 peak by Mono Q chromatography.

FIG. 7 is a chart showing an MF-3 peak by Mono Q chromatography.

FIG. 8 is a chart showing an MF-4 peak by Mono Q chromatography.

FIG. 9 is a two-dimensional electrophoretic analysis of a crude antigen2782 of Malassezia. Here, the protein is detected by staining withCoomassie brilliant blue.

FIG. 10 is a two-dimensional electrophoretic analysis of crude antigen2782 of Malassezia. Here, spots are detected by immunoblotting methodusing an IgE antibody (A) of a normal individual and an IgE antibody (B)of an allergic patient.

FIG. 11 is an electrophoretic analysis using SDS-PAGE (under reducedconditions) of MF-1, MF-2, MF-3, MF-4, and MF-13.

FIG. 12 is a graph showing the concentration dependency of the IgEbinding ability of antigenic proteins MF-1, MF-2, and MF-4.

FIG. 13 is a graph showing the concentration dependency of the IgEbinding ability of MF-3.

FIG. 14 is a chart showing purification of a pyridylethylated product ofMF-3 by HPLC.

FIG. 15 is an HPLC analytic chart of digested products oflysylendopeptidase of MF-2 (pyridylethylated product).

FIG. 16 is an HPLC analytic chart of digested products oflysylendopeptidase of MF-3 (pyridylethylated product).

FIG. 17 is comparative figures of two nucleotide sequences of MF-5 cDNA(SEQ ID NOS: 5 and 33).

FIG. 18 is comparative figures of two nucleotide sequences of MF-6 PCRfragment (SEQ ID Nos: 37 and 38).

FIG. 19 is comparative figures of nucleotide sequences of MF-1 cDNA andMF-2 cDNA (SEQ ID NOS: 1 and 2).

FIG. 20 is comparative figures of nucleotide sequences of MF-3 cDNA andMF-4 cDNA (SEQ ID NOS: 3 and 4).

FIG. 21 shows amino acid sequences of MF-1 overlapping peptides.

FIG. 22 is a graph showing the reaction between the MF-1 overlappingpeptides and RAST positive patient sera of M. furfur.

FIG. 23 is comparative figures of MF-1 cDNA and MF-1 genomic DNA (SEQ IDNOS: 18 and 19).

FIG. 24 is a chart showing MF-13 peak obtained by Phenyl Superrosechromatography.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is hereinafter described in detail.

(1) Purified Antigenic Protein of Present Invention and FunctionallyEquivalent Antigenic Proteins Thereof

The antigenic protein of the present invention is a substantially pure,isolated, antigenic protein from fungi of the genus Malassezia, whichis, in some cases, hereinafter simply referred to as “isolated andpurified antigenic protein from Malassezia” or more simply “purified,antigenic protein”, characterized in that the antigenic protein has abinding ability to IgE antibodies from patients with allergoses. Here,the phrase “substantially pure, isolated” as used herein means that theprotein of interest is substantially homogenous as a protein, whereinthe protein does not substantially contain other impurity proteins, andwherein the isolated protein is recognized as a single substance asdetermined by SDS-PAGE and isoelectric electrophoresis.

In addition, the purified, antigenic protein of the present invention ischaracterized in that the antigenic protein is a major allergen fromMalassezia reactive to patients with allergoses showing a positivereaction in a skin test to a crude antigen of Malassezia.

Also, the purified, antigenic protein of the present invention is anantigenic protein present in the fungal cells of the genus Malassezia.

Additionally, the purified, antigenic protein of the present inventionis characterized in that the antigenic protein has an epitope thereinrecognized by IgE antibodies from patients with allergoses, especiallyIgE antibodies from patients with Malassezia allergoses.

The strain which can be used in order to obtain the purified, antigenicprotein of the present invention may be any strain, as long as thestrain belongs to the genus Malassezia, and is exemplified, forinstance, by M. furfur(Malassezia furfur) TIMM2782. The above strain isidentified as Malassezia furfur TIMM2782 and deposited with an accessionnumber FERM BP-5611 with National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, which isaddressed at 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan, zipcode: 305; date of original deposit: Sep. 12, 1995; and date of transferrequest to the International Deposit: Jul. 29, 1996.

The term “major allergen from Malassezia” referred in the presentspecification is defined as a purified, antigenic protein which isrecognized by IgE antibodies, and reactive to not less than 50% of thepatients with Malassezia allergoses, i.e. patients with allergoses withpositive skin reaction to commercially available crude antigen extractsof Malassezia.

The phrase “binding ability to IgE antibodies from patients withallergoses” referred in the present specification means thatsignificantly enhanced binding, in comparison with standard sera, can beobtained, as determined by RAST method using a ¹²⁵I-labeled anti-IgEserum, or direct-RAST RIA method or ELISA method using an enzyme-labeledanti-IgE serum.

The isolated and purified, antigenic protein from Malassezia of thepresent invention has a molecular weight of from 10,000 to 100,000, asdetermined by SDS-PAGE, under reduced conditions or non-reducedconditions, and an isoelectric point of from 4 to 10 in a native stateor in a denatured state with 8 M urea, and the isolated and purified,antigenic protein from Malassezia is present in the fungal cells of thegenus Malassezia. Concrete examples thereof include MF-1, MF-2, MF-3,MF-4, MF-5, MF-6, MF-7, MF-8, MF-9, MF-10, MF-11, MF-12, MF-13, and thelike. The molecular weights, the isoelectric points, and the partialamino acid sequences of these purified, antigenic proteins will bedescribed hereinbelow.

(I) MF-1 has a molecular weight, as determined by SDS-PAGE, of about 21kDa under reduced conditions and about 40 kDa under non-reducedconditions, an isoelectric point of about 4.8 in a native state, and anisoelectric point of about 5.3 in a denatured state with 8 M urea, andcontains an amino acid sequence as shown by SEQ ID NO:45 in SequenceListing.

(II) MF-2 has a molecular weight, as determined by SDS-PAGE, of about 20kDa under reduced conditions and about 40 kDa under non-reducedconditions, an isoelectric point of about 4.8 in a native state, and anisoelectric point of about 5.8 in a denatured state with 8 M urea, andcontains amino acid sequences as shown by SEQ ID NO:46, SEQ ID NO:47,and SEQ ID NO:48, and its N-terminus is blocked.

(III) MF-3 has a molecular weight, as determined by SDS-PAGE, of about27 kDa under reduced conditions and also about 27 kDa under non-reducedconditions, an isoelectric point of about 5.2 in a native state, and anisoelectric point of about 6.5 in a denatured state with 8 M urea, andcontains amino acid sequences as shown by SEQ ID NO:49, SEQ ID NO:50,and SEQ ID NO:51, and its N-terminus is blocked.

(IV) MF-4 has a molecular weight, as determined by SDS-PAGE, of about 26kDa under reduced conditions and also about 26 kDa under non-reducedconditions, an isoelectric point of about 5.2 in a native state, and anisoelectric point of about 6.3 in a denatured state with 8 M urea, andcontains an amino acid sequence as shown by SEQ ID NO:52.

(V) MF-5 has a molecular weight, as determined by SDS-PAGE, of about 66kDa under reduced conditions, and an isoelectric point of about 6.1 in adenatured state with 8 M urea, and contains an amino acid sequence asshown by SEQ ID NO:53.

(VI) MF-6 has a molecular weight, as determined by SDS-PAGE, of about 43kDa under reduced conditions, and an isoelectric point of about 6.2 in adenatured state with 8 M urea, and contains an amino acid sequence asshown by SEQ ID NO:54.

(VII) MF-7 has a molecular weight, as determined by SDS-PAGE, of about15 kDa under reduced conditions, and an isoelectric point of about 6.0in a denatured state with 8 M urea, and contains an amino acid sequenceas shown by SEQ ID NO:55.

(VIII) MF-8 has a molecular weight, as determined by SDS-PAGE, of about30 kDa under reduced conditions, and an isoelectric point of about 5.4in a denatured state with 8 M urea, and its N-terminus is blocked.

(IX) MF-9 has a molecular weight, as determined by SDS-PAGE, of about 40kDa under reduced conditions, and an isoelectric point of about 5.3 in adenatured state with 8 M urea.

(X) MF-10 has a molecular weight, as determined by SDS-PAGE, of about 44kDa under reduced conditions, and an isoelectric point of about 6.2 in adenatured state with 8 M urea, and contains an amino acid sequence asshown by SEQ ID NO:56.

(XI) MF-11 has a molecular weight, as determined by SDS-PAGE, of about45 kDa under reduced conditions, and an isoelectric point of about 6.4in a denatured state with 8 M urea, and its N-terminus is blocked.

(XII) MF-12 has a molecular weight, as determined by SDS-PAGE, of about100 kDa under reduced conditions, and an isoelectric point of about 5.0in a denatured state with 8 M urea.

(XIII) MF-13 has a molecular weight, as determined by SDS-PAGE, of about16 kDa under reduced conditions, and an isoelectric point of about 8.1in a native state, and contains an amino acid sequence as shown by SEQID NO:57.

The isolated and purified, antigenic protein from Malassezia of thepresent invention may be any protein, as long as the antigenic proteinis from Malassezia and recognized as an antigen of mammals, includinghumans, and the antigenic protein is not limited to the 13 kinds ofpurified, antigenic proteins exemplified above.

Furthermore, diagnoses using these purified, antigenic proteins yieldresults correlating to those of diagnoses based on skin tests and RASTmethod using extracts of crude conventional antigen of Malassezia.Specifically, many of the patients showing positive reaction in a skintest using crude antigens also show positive reaction for IgE antibodytiter against the crude antigens of Malassezia. Not less than 50% of thepatients with positive reaction for IgE antibody titer against crudeantigens have high IgE antibody titers against the above-describedisolated and purified, antigenic protein of the present invention (seeTables 2 and 3 in Examples set forth below).

Also, when administered to patients with Malassezia allergoses, thepurified, antigenic protein of the present invention is capable oflowering the allergic response to Malassezia fungi in patients withMalassezia allergoses administered therewith.

Moreover, the present invention provides functionally equivalentantigenic proteins having properties immunologically equivalent to thoseof the above-described purified, antigenic protein. For example, asfunctional equivalents having properties immunologically equivalent tothose of the above-described 13 kinds of purified, antigenic proteins,functional equivalents of various strains of M. furfur, and functionalequivalents of fungal species of the genus Malassezia other than M.furfur, are also encompassed in the scope of the present invention.Specifically, MF-2 is homologous to a peroxisome membrane protein PMP-20[L. Garrard et al., J. Biol. Chem., 23, 13929-13937 (1989)], andproteins from Malassezia having similar immunological properties areencompassed in the scope of the present invention. Also, MF-3 and MF-4,which are different proteins, are both homologous toiron/manganese-superoxide dismutase [T. Matsumoto et al., Biochemistry,30, 3210-3216 (1991); M. L. Ludwig et al., J. Mol. Biol., 219, 335-358(1991)]; and MF-5, MF-6, and MF-13 are homologous to dihydrolipoamidedehydrogenase (DLDH), malate dehydrogenase (MDH), and cyclophilin,respectively, and proteins from Malassezia having similar immunologicalproperties are encompassed in the scope of the present invention.

Incidentally, the purified, antigenic protein of the present inventioncan be modified, derivatized, or bound to polyethylene glycol (PEG) bythe PEG method [Wie et al., Int. Arch. Allergy Appl. Immunol., 64, 84-99(1981)], in order to enhance stability and/or desired reactivity, i.e.to enhance antigen-antibody specific binding for diagnostic purposes, orto attenuate allergic reaction or eliminate enzymatic activity fortherapeutic purposes. Protein modifications include pyridylethylation,reduction, alkylation, acylation, chemical coupling to suitablecarriers, gentle formalin treatment, and guanidine hydrochloridetreatment.

(2) Antigenic Fragment of Present Invention

The antigenic fragment of the present invention is an antigenic fragmentderived from the purified, antigenic protein, characterized in that theantigenic protein has an antigenic epitope contained in theabove-described purified, antigenic protein. The antigenic fragments areexemplified by, for instance, antigenic fragments derived from purified,antigenic protein containing at least one antigenic epitope contained inMF-1, MF-2, MF-3, MF-4, MF-5, MF-6, MF-7, MF-8, MF-9, MF-10, MF-11,MF-12, MF-13, and the like, among which preference is given to thosecontaining at least one T cell epitope or B cell epitope. The antigenicfragments of the present invention include fragments derived from thepurified, antigenic protein of Malassezia. The fragments cause immuneresponses in mammals, especially in humans, for instance, minimum levelsof stimulation of IgE production, IgE binding, induction of IgG and IgMantibody production, and T cell proliferation, and/or lymphokinesecretion, and/or induction of T cell anergy.

When using the antigenic fragment of the present invention fortherapeutic purposes, it is desired that the antigenic fragment is weakin activation of T cell response, or induces T cell anergy. Also, it ispreferred that the antigenic fragment of the present invention does notsubstantially have a binding ability to IgE antibodies specific toMalassezia fungi, or even when the antigenic fragment is bound to theIgE antibody, the binding is at a level where no mediators, such ashistamine, are released from mast cells or basophiles. In other words,it is preferred that even when binding to IgE antibodies occurs, theantigenic fragment binds to IgE antibodies at levels substantially lowerthan those for the purified, antigenic proteins from Malassezia. Asdescribed above, the antigenic fragment of the present inventionpreferably has a lower activity of activation in IgE-mediated immuneresponse than that of the purified, antigenic proteins when used fortherapeutic purposes. Therefore, when administered to patients withMalassezia allergoses, it is made possible to reduce allergic responsesto Malassezia fungi in patients with Malassezia allergoses administeredtherewith.

The antigenicity of the antigenic fragment of the present invention canalso be assessed in in vitro tests, such as RAST method, ELISA method,and histamine release tests, as well as in skin tests and intracutaneoustests to human volunteers.

The term “epitope” is a basic element or minimum unit recognized byreceptors, especially antibodies, such as immunoglobulins,histocompatibility antigens, and T cell receptors, and contains aminoacid sequences essential for receptor recognition. Other peptidesresembling the amino acid sequence of an epitope, which can lower theallergic response to a Malassezia allergen, can also be used asepitopes. It is possible to design a Malassezia allergen peptide whichis likely to change the allergic response to Malassezia fungi inpatients with Malassezia allergoses when administered in sufficientamounts to the patients by currently available information on proteinstructures. It is also possible to design reagents or drugs whichinhibit induction of allergic reaction in patients with Malasseziaallergoses. For example, such drugs can be designed to bind to IgEantibodies against Malassezia allergens, and to thereby interfere withIgE-allergen binding and subsequent degranulation from mast cells.

Also, selection of peptides containing a T cell epitope can be carriedout by culturing T lymphocytes obtained from an individual sensitive toa Malassezia allergen, i.e. individuals with IgE-mediated immuneresponse, with a peptide from allergen, and then measuring stimulatingactivity for human T cell, i.e. blast formation activity, for instance,by means of determining whether or not T cell proliferation occurs inresponse to the addition of the peptide by measuring incorporation oftritiated thymidine into cells. Peptides containing a B cell epitope canbe selected by reacting sera obtained from an individual sensitive to aMalassezia allergen with each peptide derived from the allergen, andmeasuring the amount of bound IgE to the peptide.

Peptides having immunological cross-reactivity to the fragment of thepurified, antigenic proteins from Malassezia, including Malasseziaallergens, for instance, those recognized by specific antibodies or Tcells against the fragment thereof are encompassed in the antigenicfragment of the present invention.

In order to prepare the antigenic fragment of the present invention, anisolated and purified, antigenic protein, a starting material, isenzymatically digested with a protease, such as lysylendopeptidase ortrypsin, or cleaved by chemical treatment with agents such as cyanogenbromide, after which a fragment having a desired antigenicity isisolated and purified by known methods of protein purification. It isalso possible to express and prepare the desired antigenic fragmentusing a portion of the gene encoding an antigenic protein derived fromMalassezia. Further, it can be also prepared by chemical synthesisutilizing peptide synthesis technology based on information on thechemical structure of the antigenic fragment.

In addition, amino acid substitution, insertion and deletion can becarried out using genetic engineering techniques and chemical synthesistechniques. For example, to enhance stability and/or enhance the desiredreactivity, the antigenic fragment of the present invention may bederivatized, or modified by deletion, insertion, substitution oraddition of at least one amino acid. The modified protein or peptide ofthe present invention can also be modified by replacing an amino acidwith a D-amino acid, a non-natural amino acid, or a non-natural aminoacid analogue, or by adding these amino acids or analogues. Theantigenic fragment of the present invention can also be chemicallymodified by binding with polyethylene glycol. Modifications of theantigenic fragment include reduction, alkylation, acylation, andchemical coupling to suitable carriers.

The antigenic fragment thus obtained can be determined and isolated bymeasuring the induction of immune responses, including activation of Tcell response, induction of T cell anergy, binding with antibody, andthe like.

Next, the method for producing the purified, antigenic protein of thepresent invention will be described below. Conventionally used crudeantigens have been lyophilized products of culture filtrates, orpurified products obtained from cultured cells by very limited means ofpurification, such as disrupting the cells by a suitable method toobtain an extract, and then subjected to precipitation with ammoniumsulfate and lyophilizing. The present inventors have also attemptedpurification using such crude antigens as starting materials by commonlyused methods of protein purification, e.g., gel filtration, ion exchangeand other chromatographies, but they have not succeeded in isolation ofa single pure, antigenic protein using these techniques only.

The isolated and purified, antigenic protein from Malassezia of thepresent invention can be isolated by fractionating a crude antigenprepared from Malassezia cells as a starting material by an appropriatecombination of effective separation methods using ion exchangechromatography, chelate resin chromatography, hydrophobicchromatography, gel filtration chromatography, and the like, thenmeasuring the binding of each fraction with an IgE antibody of patientsera by RAST method, immunoblotting, and the like, to search for aprotein that binds to the IgE antibody in the allergic patient sera, orto search for a protein that induces immune responses, includingactivation of T cell response, T cell anergy, and the like, by variousmethods using patient lymphocytes.

Specifically, a fungus of the genus Malassezia, such as M. furfur, iscultured under appropriate temperature, aeration and other conditionsusing a medium containing nutrients suitable for the growth ofMalassezia fungi, supplemented with olive oil or Tween 40 or Tween 60,such as Dixon medium. The obtained cells are disrupted by a suitablemethod to yield an extract. From this extract, the antigenic protein canbe purified using separation means, including ion exchangechromatography, chelate resin chromatography, and hydrophobicchromatography. In other words, the antigenic protein can be isolated asa high-purity protein using an appropriate combination of various knownmethods of peptide and protein purification, such as ion exchangechromatography, hydrophobic chromatography, gel filtrationchromatography, chelate resin chromatography, electrophoresis, andaffinity chromatography using a resin coupled with an antibody specificto an antigenic protein derived from Malassezia or an antigenic fragmentthereof. The antigenic protein contained in the culture filtrate can beisolated in the same manner.

Specifically, as shown in Examples below, a group of a large number ofwell-resembled proteins that are inseparable on the basis of molecularweight can be separated from each other by combining ion exchangechromatography, utilizing the differences in isoelectric points;hydrophobic chromatography, utilizing differences in hydrophobicity;chelate resin chromatography, utilizing differences in chelatingabilities with metals; gel filtration chromatography, utilizing themolecular weight differences, and the like. These findings have beenunexpected from the findings concerning differences of the antigenicproteins on the basis of the molecular weight shown by conventionalSDS-PAGE immunoblotting. For example, MF-1 and MF-2 are almost identicalin terms of molecular weight, and they are mutually inseparable byconventional SDS-PAGE. It is also impossible to mutually separate MF-3and MF-4 on the basis of molecular weight.

Concrete examples of the combinations of various separation means aregiven below, as exemplified by the following steps:

Step a: Centrifuging a cell disruption extract of a cultured Malasseziafungus, lyophilizing the resulting supernatant, and thereaftersubjecting the lyophilized product to anionic exchange chromatography(for instance, DEAE-cellulose column chromatography, manufactured byWako Pure Chemical Industries) to obtain a fraction eluted with 0.1 MNaCl;

Step b: Concentrating the eluted fraction obtainable in Step a using anultrafiltration membrane (MW 10,000), and thereafter subjecting theresulting concentrate to gel filtration chromatography (for instance,Sephacryl S-200HR column chromatography, manufactured by Pharmacia) toobtain a fraction eluted at molecular weights of 30,000 to 50,000;

Step c: Concentrating the eluted fraction obtainable in Step b using anultrafiltration membrane (MW 10,000), and thereafter subjecting theconcentrate to gel filtration chromatography (for instance, SephadexG-75 Superfine column chromatography, manufactured by Pharmacia) toobtain a fraction eluted at a molecular weight of about 40,000;

Step d: Subjecting the eluted fraction obtainable in Step c to zincchelating chromatography (for instance, Zinc Chelating Sepharose fastflow column chromatography, manufactured by Pharmacia), and furthersubjecting the resulting effluent fraction to copper chelatechromatography to obtain an effluent fraction or a fraction eluted at pHabout 4;

Step e: Concentrating the effluent fraction or the fraction eluted at pHabout 4 obtainable in Step d, and thereafter purifying the resultingconcentrate by gel filtration chromatography (for instance, SephadexG-75 Superfine column chromatography, manufactured by Pharmacia) toobtain a fraction eluted at a molecular weight of about 40,000; and

Step f: Further purifying the eluted fraction obtainable in Step e byion exchange chromatography of Mono Q.

Alternatively, there may be included the following steps as one example.

Step a: Centrifuging a cell disruption extract of a cultured Malasseziafungus; lyophilizing the resulting supernatant, and thereaftersubjecting the lyophilized product to anionic exchange chromatography(for instance, DEAE-cellulose column chromatography) to obtain afraction eluted with 0.1 M NaCl;

Step b: Concentrating the eluted fraction obtainable in Step a using anultrafiltration membrane (MW 10,000), and thereafter subjecting theresulting concentrate to gel filtration chromatography (for instance,Sephacryl S-200HR column chromatography) to obtain a fraction eluted atmolecular weights of 30,000 to 50,000;

Step c: Concentrating the eluted fraction obtainable in Step b using anultrafiltration membrane (MW 10,000), and thereafter subjecting theresulting concentrate to gel filtration chromatography (for instance,Sephadex G-75 Superfine column chromatography) to obtain a fractioneluted at a molecular weight of about 40,000;

Step d: Subjecting the eluted fraction obtainable in Step c to zincchelating chromatography (for instance, Zinc Chelating Sepharose fastflow column chromatography) to obtain a fraction eluted at pH about 5;and

Step g: Concentrating the eluted fraction obtainable in Step d, andthereafter purifying the resulting concentrate by subjecting theconcentrate to gel filtration chromatography (for instance, SephadexG-75 Superfine column chromatography).

Next, the method of the present invention will be explained in furtherdetail by taking, as examples, the production methods for purified,antigenic proteins (MF-1, MF-2, MF-3, MF-4, and MF-13) of the presentinvention. However, the following steps are simply examples, withoutintending to limit the scope of the present invention thereto.

1. Production Example of MF-1

This method comprises centrifuging a cell disruption extract of culturedM. furfur (Malassezia furfur) TIMM 2782 cells, lyophilizing theresulting supernatant, and thereafter subjecting the lyophilized productto anionic exchange chromatography (for instance, DEAE-cellulose columnchromatography) to obtain a fraction eluted with 0.1 M NaCl;concentrating the resulting eluted fraction using an ultrafiltrationmembrane (MW 10,000), and thereafter subjecting the resultingconcentrate to gel filtration chromatography (for instance, SephacrylS-200HR column chromatography) to obtain a fraction eluted at molecularweights of 30,000 to 50,000; concentrating the resulting eluted fractionusing an ultrafiltration membrane (MW 10,000), and thereafter subjectingthe resulting concentrate to gel filtration chromatography (forinstance, Sephadex G-75 Superfine column chromatography) to obtain afraction eluted at a molecular weight of about 40,000; subjecting theresulting eluted fraction to zinc chelating chromatography (forinstance, Zinc Chelating Sepharose fast flow column chromatography), andfurther subjecting the resulting effluent fraction to copper chelatechromatography to obtain a fraction eluted at a pH of about 4; andconcentrating the resulting eluted fraction, and thereafter purifyingthe concentrate by gel filtration chromatography (for instance, SephadexG-75 Superfine column chromatography) to obtain a fraction eluted at amolecular weight of about 40,000.

2. Production Example MF-2

This method comprises centrifuging a cell disruption extract of culturedM. furfur (Malassezia furfur) TIMM 2782 cells, lyophilizing theresulting supernatant, and thereafter subjecting the lyophilized productto anionic exchange chromatography (for instance, DEAE-cellulose columnchromatography) to obtain a fraction eluted with 0.1 M NaCl;concentrating the resulting eluted fraction using an ultrafiltrationmembrane (MW 10,000), and thereafter subjecting the resultingconcentrate to gel filtration chromatography (for instance, SephacrylS-200HR column chromatography) to obtain a fraction eluted at molecularweights of 30,000 to 50,000; concentrating the resulting eluted fractionusing an ultrafiltration membrane (MW 10,000), and thereafter subjectingthe resulting concentrate to gel filtration chromatography (forinstance, Sephadex G-75 Superfine column chromatography) to obtain afraction eluted at a molecular weight of about 40,000; subjecting theresulting eluted fraction to zinc chelating chromatography (forinstance, Zinc Chelating Sepharose fast flow column chromatography) toobtain a fraction eluted at a pH of about 5; and concentrating theresulting eluted fraction, and thereafter purifying the resultingconcentrate by gel filtration chromatography (for instance, SephadexG-75 Superfine column chromatography).

3. Production Example MF-3

This method comprises centrifuging a cell disruption extract of culturedM. furfur (Malassezia furfur) TIMM 2782 cells, lyophilizing theresulting supernatant, and thereafter subjecting the lyophilized productto anionic exchange chromatography (for instance, DEAE-cellulose columnchromatography) to obtain a fraction eluted with 0.1 M NaCl;concentrating the resulting eluted fraction using an ultrafiltrationmembrane (MW 10,000), and thereafter subjecting the resultingconcentrate to gel filtration chromatography (for instance, SephacrylS-200HR column chromatography) to obtain a fraction eluted at molecularweights of 30,000 to 50,000; concentrating the resulting eluted fractionusing an ultrafiltration membrane (MW 10,000), and thereafter subjectingthe resulting concentrate to gel filtration chromatography (forinstance, Sephadex G-75 Superfine column chromatography) to obtain afraction eluted at a molecular weight of about 40,000; subjecting theresulting eluted fraction to zinc chelating chromatography (forinstance, Zinc Chelating Sepharose fast flow column chromatography) toobtain an effluent fraction, and further subjecting the effluentfraction to copper chelate chromatography; concentrating the resultingeffluent fraction, and thereafter purifying the resulting concentrate bygel filtration chromatography (for instance, Sephadex G-75 Superfinecolumn chromatography) to obtain a fraction eluted at a molecular weightof about 40,000; and further purifying the resulting fraction by anionicexchange chromatography of Mono Q.

4. Production Example MF-4

This method comprises centrifuging a cell disruption extract of culturedM. furfur (Malassezia furfur) TIMM 2782 cells, lyophilizing theresulting supernatant, and thereafter subjecting the lyophilized productto anionic exchange chromatography (for instance, DEAE-cellulose columnchromatography) to obtain a fraction eluted with 0.1 M NaCl;concentrating the resulting eluted fraction using an ultrafiltrationmembrane (MW 10,000), and thereafter subjecting the resultingconcentrate to gel filtration chromatography (for instance, SephacrylS-200HR column chromatography) to obtain a fraction eluted at molecularweights of 30,000 to 50,000; concentrating the resulting eluted fractionusing an ultrafiltration membrane (MW 10,000), and thereafter subjectingthe resulting concentrate to gel filtration chromatography (forinstance, Sephadex G-75 Superfine column chromatography) to obtain afraction eluted at a molecular weight of about 40,000; subjecting theresulting eluted fraction to zinc chelating chromatography (forinstance, Zinc Chelating Sepharose fast flow column chromatography) toobtain an effluent fraction, and further subjecting the effluentfraction to copper chelate chromatography; concentrating the resultingeffluent fraction, and thereafter purifying the resulting concentrate bygel filtration chromatography (for instance, Sephadex G-75 Superfinecolumn chromatography) to obtain a fraction eluted at a molecular weightof about 40,000; and further purifying the resulting fraction by anionicexchange chromatography of Mono Q.

5. Product Example MF-13

This method comprises centrifuging a cell disruption extract of culturedM. furfur (Malassezia furfur) TIMM 2782 cells, lyophilizing theresulting supernatant, and thereafter subjecting the lyophilized productto anionic exchange chromatography (for instance, DEAE-cellulose columnchromatography) to collect a non-adsorbing fraction; subjecting thefraction to gel filtration chromatography (for instance, Superdex 75 pg)to obtain an eluted fraction with a molecular weight of not more than20,000; subjecting the resulting fraction to SP cationic exchangechromatography to obtain a fraction eluted with 0.2 M NaCl; and furtherpurifying the eluted fraction by gel filtration chromatography (forinstance, Superdex 75 pg).

In addition, the antigenic protein derived from Malassezia of thepresent invention can be prepared as a recombinant protein by a methodcomprising isolating a gene encoding the protein by such methods as PCRbased on the information on the amino acid sequence mentioned above, andinserting the genes into a vector by genetic engineering techniques soas to be expressed in E. coli, yeasts, molds, mammalian cells, and thelike.

(3) Antibody or Antibody Fragment of Present Invention Against Purified,Antigenic Protein or Antigenic Fragment Thereof

The antibody of the present invention against an isolated and purified,antigenic protein from Malassezia or an antigenic fragment thereof canbe prepared by using as an antigen the purified, antigenic protein fromMalassezia of the present invention, an antigenic fragment obtainable byenzymatic or chemical treatment of the above protein, or an antigenicpeptide obtained by chemical synthesis. The antibody can be prepared bya conventional method including, e.g., a method comprising immunizing ananimal, such as a rabbit, with the above-described protein or a fragmentthereof together with an adjuvant to obtain an antiserum. Also, amonoclonal antibody can be prepared by fusing an antibody-producing Bcell obtainable by immunizing an antigen and a myeloma cell, selecting ahybridoma for producing the desired antibody, and culturing this cell.These antibodies can be used for production of an antigenic protein,measurement of titration of antigen extract of Malassezia allergen, andother purposes, as described later. As hybridomas mentioned above, ahybridoma for producing an M-40 monoclonal antibody against theantigenic protein MF-1 is named and identified as 5B4; a hybridoma forproducing an M-3 monoclonal antibody against the antigenic protein MF-2is named and identified as 8G11; and hybridoma for producing an M-1monoclonal antibody against the against the antigenic protein MF-3 isnamed and identified as 10C1, and these hybridomas are deposited as FERMBP-5608, FERM BP-5609, and FERM BP-5610, respectively, with NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, addressed at 1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan (zip code: 305; date of original deposit: Sep. 12,1995; date of transfer request to the International Deposit: Jul. 29,1996.

(4) Diagnostic Agent of Present Invention Containing as ActiveIngredient Purified, Antigenic Protein or Antigenic Fragment Thereof

The present invention provides a diagnostic agent for allergoses orinfectious diseases of which causative microorganisms are Malasseziafungi, using an isolated and purified, antigenic protein from Malasseziaor an antigenic fragment having at least one antigenic epitope derivedfrom the antigenic protein.

The term “allergoses of which causative microorganisms are Malasseziafungi” as used herein is defined as any allergoses of which causativemicroorganisms are Malassezia fungi, exemplified by atopic bronchialasthma, allergic rhinitis, allergic conjunctivitis, and atopicdermatitis. The term “infectious disease of which causativemicroorganisms are Malassezia fungi” is defined as any infectiousdisease of which causative microorganisms are Malassezia fungi,exemplified by tinea versicolor, Malassezia folliculitis, and dandruff.

The diagnostic agent for allergoses of the present invention is used asan intracutaneous reaction diagnostic agent and titration reagent forallergy diagnosis in allergoses caused by Malassezia fungi. When used asan intracutaneous reaction diagnostic agent, the isolated and purified,antigenic protein of the present invention or the antigenic fragment ofthe present invention is dissolved in a buffer and diluted inphenol-containing physiological saline by a conventional method.

Also, when used as a titration reagent for allergy diagnosis, it can beprepared by a conventional method. For example, the isolated andpurified, antigenic protein of the present invention or the antigenicfragment of the present invention may be suitably dissolved and dilutedin a Hanks' buffer to be used as a histamine release titration reagent.The method can be usually carried out by the following procedures.Specifically, a given volume of blood of a patient with allergoses or agiven number of blood cells prepared by suspending a fraction of bloodcells obtained by centrifugation is titrated with a solution of thementioned purified, antigenic protein as a titration reagent bymeasuring the amount of histamine, which is released from basophiles,upon allergen stimulation by HPLC.

The isolated and purified, antigenic protein of the present invention orthe antigenic fragment of the present invention can also be used fordetection and diagnosis of Malassezia allergoses. For example, thediagnosis can be carried out by incubating blood or a blood componentsampled from a patient whose sensitivity to Malassezia fungi is toassessed, together with the isolated and purified, antigenic protein ofthe present invention, and the like under appropriate conditions, anddetermining the degree of binding of the purified, antigenic proteinwith a blood component, including, for instance, antibody, T cell, Bcell, or the like.

(5) Therapeutic Drug of Present Invention Containing as ActiveIngredient Purified, Antigenic Protein or Antigenic Fragment Thereof

The present invention provides a therapeutic drug for allergoses ofwhich causative microorganisms are Malassezia fungi, including, as anactive ingredient, an isolated and purified, antigenic protein fromMalassezia or an antigenic fragment having at least one antigenicepitope.

The therapeutic drug of the present invention for allergoses can beadministered via ordinary pathways, including, for instance, oral,intracutaneous, subcutaneous, intramuscular, and intraperitonealpathways. Further, it can be used as percutaneous or transmucosal drugs,such as troches, sublingual tablets, eyedrops, intranasal sprays,poultices, creams, and lotions. Regarding the dosage and administrationfrequency of the therapeutic drug of the present invention forallergoses, the therapeutic drug can be suitably administered at aselected dose in a range of about not more than 20 mg per administrationfor an adult, depending on administration pathways, symptoms, and thelike, and about once every week. Also, the therapeutic drug of thepresent invention for allergoses is useful not only as a therapeuticdrug but also as a prophylactic drug for allergoses caused by Malasseziafungi. This is because it exhibits little or no anaphylaxis-inducingaction and thus can be used safely in humans.

The therapeutic drug of the present invention for Malassezia allergosescontains as an active ingredient the above-described purified, antigenicprotein or an antigenic fragment thereof, and is used as a therapeuticdrug and prophylactic drug for various allergoses caused by Malasseziafungi.

The method of preparing the therapeutic drug of the present inventionfor allergoses is not particularly limited. For example, the purified,antigenic protein of the present invention or an antigenic fragmentthereof having an epitope may be dried to a powder form and used as ahyposensitization therapeutic drug for allergoses caused by Malasseziafungi. In this case, it can be used alone, or in the form of acombination drug containing commonly used adjuvants and variousadditives, such as stabilizers, excipients, dissolution aids,emulsifiers, buffers, soothing agents, preservatives, and coloringagents, which are added by conventional methods as occasion demands. Forexample, a purified, antigenic protein in the powder form is dissolvedin a phenol-supplemented physiological saline and used for a stocksolution of an antigen for hyposensitization treatment.

In order to use it as a hyposensitization therapeutic drug, it isparticularly advantageous that the therapeutic agent has an epitope thatdoes not bind to IgE specific to Malassezia fungi, or even when theantigenic fragment is bound to the IgE, the binding is at a level whereno histamine is released from mast cells or basophiles.

(6) Method for Quantitative Assay of Malassezia Allergen

The present invention also provides a method for quantitative assay ofthe Malassezia allergen. The antibody against the purified, antigenicprotein from Malassezia can be used for an immunological quantitativeanalysis of the Malassezia allergen usable in diagnoses of allergoses orinfectious diseases of which causative microorganisms are Malasseziafungi.

It is easy to establish a method for quantitative assay by such methodsas ELISA, using, the isolated and purified, antigenic protein of thepresent invention or the recombinant antigenic protein descried later asa standard allergen and the antibody against the antigenic protein. SomeMalassezia antigen extracts are commercially available, as describedabove. Also, because Malassezia fungi are commonly present on skins,including the human scalp, it is thought that commercially availablehouse dust samples contain Malassezia allergens. It is extremely usefulfrom diagnostic and therapeutic viewpoints to make known the Malasseziaallergen contents in these commercially available antigen extracts.

(7) Recombinant Malassezia Antigenic Protein

The present invention provides a recombinant Malassezia antigenicprotein (hereinafter, simply abbreviated as “recombinant antigenicprotein” in some cases) having immunological properties equivalent tothose of the pure, isolated and purified antigenic protein fromMalassezia of Item (1) above, the purified, antigenic protein having abinding ability to an IgE antibody from patients with allergoses.Examples thereof include, for instance, a group of peptides comprisingrMF-1 to -7 having amino acid sequences as shown by any one of SEQ IDNOs:8 to 14 (here, the term “rMF-1 to -7” means MF-1 to -7 obtained bymeans of a genetic recombination method), and having immunologicalproperties equivalent to those of the above peptides. Specifically,there are included in the present invention peptides having an entire orpartial amino acid sequence as shown by any one of SEQ ID NOs:8 to 14;peptides including the above peptides having immunological propertiesequivalent to those of each of MF-1 to -7 corresponding to rMF-1 to -7;and peptides comprising amino acid sequences, wherein the antigenicprotein results from at least one of deletion, addition, insertion orsubstitution of one or more amino acid residues in the amino acidsequence as shown by any one of SEQ ID NOs:8 to 14, or a partialsequence thereof, wherein the antigenic protein has immunologicalproperties equivalent to those of each of MF-1 to -7 corresponding torMF-1 to -7.

For instance, in a case where rMF-1 is taken as an example, rMF-1includes peptides which are antigenic proteins having immunologicalproperties equivalent to those of MF-1, and having an entire or partialamino acid sequence as shown by SEQ ID NO:8 in Sequence Listing, orrecombinant Malassezia antigenic proteins including the above peptide.Further, rMF-1 includes recombinant Malassezia antigenic proteinswherein the antigenic protein results from at least one of deletion,addition, insertion or substitution of one or more amino acid residuesin the amino acid sequence as shown by SEQ ID NO:8 in Sequence Listing,or a partial sequence thereof, wherein the antigenic protein hasimmunological properties equivalent to those of each of MF-1corresponding to rMF-1. The same can be said for rMF-2 to -7.

Here, the phrase “immunological properties equivalent” refers to thosehaving equivalent Malassezia allergen activity, and the term “Malasseziaallergen activity” refers to a binding ability to IgE antibodies frompatients with allergoses, especially those with Malassezia allergoses.

The recombinant Malassezia antigenic protein of the present invention isobtained by, as a recombinant protein, selecting an appropriate vectorso as to express the protein in bacteria, such as Escherichia coli,yeasts, such as budding yeasts, fungi, such as Aspergillus, insectcells, mammalian cells, and the like, by genetic engineering techniquesusing the gene of the present invention described later, preparing anexpression vector, and introducing it into the above cells. Therecombinant Malassezia antigenic protein is, therefore, essentially freeof other proteins from Malassezia.

Functional equivalents to the recombinant antigenic protein of thepresent invention may be obtained by modifying the recombinant antigenicprotein by known methods using mutagenesis in a specific site of the DNAencoding the recombinant antigenic protein of the present invention. Forexample, substitution, insertion, deletion or addition of one or morebases on the polynucleotide described later enables to makesubstitution, insertion, deletion or addition of an amino acid residue.It is also possible to select a mutant retaining the biologicalactivity.

Known methods of preparing the above mutants include a gapped duplexmethod [Nucleic Acids Research, 12, 24, 9441-9456 (1984)], a deletionmethod [Gene, 33, 103-119 (1985)], a PCR method [Gene, 102, 67-70(1991)], uracil DNA methods [Methods in Enzymology, 154, 367-382 (1987);Proc. Natl. Acad. Sci. USA, 79, 7258-7262 (1982)], and a cassettemutation method [Gene, 24, 315-323 (1985)].

A tag group may be added to the peptide chain to facilitate thepurification of the recombinant antigenic protein of the presentinvention or to increase its solubility. An example of the tag groupincludes polyhistidine, which can be purified by metal affinitychromatography. Additionally, if necessary, an endoprotease-specificrecognition site may be introduced between the tag group and the desiredpeptide, and the resulting peptide is then treated with the protease, tofacilitate the isolation of the peptide free of undesirable sequences.

In order to succeed in desensitization of a patient to a peptideantigen, it is necessary to increase the solubility of the peptide byadding a functional group to the peptide, or by not including ahydrophobic T cell epitope, a hydrophobic epitope, or a hydrophobicregion in the peptide. Also, in order to aid appropriate antigenprocessing of the T cell epitope in the peptide antigen, an endoproteaserecognition site may be prepared between two regions each containing atleast one T cell epitope by the above-described recombination techniqueor synthesis. For example, a charged amino acid pair, such as LysLys orArgArg, may be introduced between such regions within the peptide, andthe resulting peptide is sensitive to cleavage with cathepsin and/orother trypsin-like enzymes, permitting production of a peptide fragmentcontaining 1 or more T cell epitopes. In the addition, the charged aminoacid residues as described above are also capable of increasing peptidesolubility.

(8) Polynucleotide Encoding Recombinant Malassezia Antigenic Protein ofPresent Invention

The present invention provides a polynucleotide encoding the recombinantMalassezia antigenic protein, or a polynucleotide encoding antigenicfragments thereof. The polynucleotides include polynucleotides eachhaving an entire or partial sequence of the base sequence as shown byany one of SEQ ID NOs:1 to 7 in Sequence Listing, or a polynucleotidecontaining the polynucleotide, wherein each of the polynucleotideencoding rMF-1 to -7 or an antigenic protein having immunologicalproperties equivalent to these proteins. In addition, there are alsoincluded polynucleotides encoding the recombinant Malassezia antigenicprotein, wherein the polynucleotide results from at least one ofdeletion, addition, insertion or substitution of one or more bases inthe base sequence having an entire or partial sequence of the basesequence as shown by any one of SEQ ID NOs:1 to 7 in Sequence Listing.Further, there are included polynucleotides capable of hybridizing tothe polynucleotide, wherein the polynucleotides each encodes anantigenic protein having Malassezia allergen activity.

For instance, in a case where rMF-1 is taken as an example, there areencompassed in the present invention polynucleotides each having anentire sequence of the base sequence as shown by SEQ ID NO:1 in SequenceListing, or a partial sequence thereof, or a polynucleotide containingthe polynucleotide, wherein each of the polynucleotide encoding rMF-1 oran antigenic protein having immunological properties equivalent to theprotein. In addition, there are also encompassed in the presentinvention polynucleotides encoding the recombinant Malassezia antigenicprotein, wherein the antigenic protein results from at least one ofdeletion, addition, insertion or substitution of one or more bases in abase sequence comprising an entire sequence as shown by SEQ ID NO:1 inSequence Listing, or a partial sequence thereof. Further, there areincluded polynucleotides capable of hybridizing to the polynucleotide,wherein the polynucleotides each encodes an antigenic protein havingMalassezia allergen activity. The same can be said for rMF-2 to -7.

The polynucleotide encoding a recombinant Malassezia antigenic proteincan be obtained by a method as described below. It is possible todetermine the N-terminal amino acid sequence or internal amino acidsequence of a Malassezia antigenic protein purified by a combination ofvarious ordinary chromatographies, or that of a Malassezia antigenicprotein purified by one-dimensional or two-dimensional electrophoresis.An oligonucleotide capable of encoding these amino acid sequences issynthesized and purified. Since one kind of amino acid is usuallyencoded by a number of codons, this oligonucleotide is a mixtureprepared in consideration of all these codons. PCR is carried out toyield a polynucleotide of the present invention encoding the Malasseziaantigenic protein, using this oligonucleotide and oligo(dT) as primers,and a cDNA synthesized from a total RNA or a genomic DNA extracted andpurified from Malassezia fungi as a template. Oligonucleotidescorresponding to two portions of an amino acid sequence for theantigenic protein may be used as primers for PCR, and PCR may berepeated in cases when the cDNA is not amplified by carrying out PCRonce.

A polynucleotide containing the entire sequence or a polynucleotidecapable of hybridizing to a polynucleotide encoding antigenic proteincan easily be obtained by screening a cDNA library or genomic DNAlibrary prepared from the poly(A)⁺ RNA or genomic DNA of Malasseziafungi, using the cDNA fragment obtained by the PCR reaction as a probefor DNA hybridization. The vector used for library preparation may be ofphage origin or plasmid origin.

As another method, a cDNA clone encoding a Malassezia antigenic proteinpossessing Malassezia allergen activity can be obtained by preparing acDNA expression library prepared from a poly(A)⁺ RNA of Malasseziafungi, and screening a clone producing the proteins that binds to theIgE antibody derived from a patient with allergoses. The proteinexpressed by this cDNA clone is a Malassezia antigenic protein.

The genes encoding the epitopes from Malassezia described below are alsoencompassed in the present invention, having sequences with a lessnumber of bases than those in the base sequence encoding the entireamino acid sequence of a Malassezia allergen. Generally, although thebase sequence encoding an epitope is selected from base sequencesencoding mature proteins, in some cases, it is desired that a basesequence is selected to contain the leader sequence portion of thepresent invention. The gene of the present invention may contain alinker sequence containing a restriction endonuclease recognition siteand/or a sequence useful for the cloning, expression, or purification ofthe desired gene. Specifically, there are encompassed in the presentinvention polynucleotides encoding at least one B cell epitope andhaving a partial sequence of any one of the base sequences shown by SEQID NOs:1 to 7, or polynucleotides resulting from partial modificationsthereof by chemical or physical methods. For example, there are alsoencompassed in the present invention the corresponding polynucleotidespossessed by M. furfur strains other than the strain used in the presentinvention or other fungi of the genus Malassezia, for example, M.pachydermatis and M. sympodialis. Specifically, M. furfur can beclassified into five groups according to physiological properties(“Japanese Journal of Medical Mycology,” Katsuhisa Uchida), each havinga corresponding gene, and these genes are also encompassed in thepresent invention.

Moreover, the present invention includes polynucleotides capable ofhybridizing to a polynucleotide having a base sequences shown by any oneof SEQ ID NOs:1 to 7, or a base sequence encoding at least one B cellepitope. In the present invention, the term “capable of hybridizing”refers to a polynucleotide capable of hybridizing to anotherpolynucleotide under the conditions shown below. A membrane on which DNAis immobilized is incubated with a probe at 50° C. for 12 to 20 hours in6×SSC (1×SSC showing 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0)containing 0.5% SDS, 0.1% bovine serum albumin (BSA), 0.1%polyvinylpyrrolidone, 0.1% Ficol 400, and 0.01% denatured salmon spermDNA. After termination of the incubation, the membrane is washed untilthe signal from the immobilized DNA becomes distinguishable from thebackground firstly at 37° C. in 2×SSC containing 0.5% SDS, wherein theSSC concentration is changed to 0.1 fold the starting level, and whereinthe temperature is changed to 50° C., and then the detection with aprobe is carried out. By examining the activity owned by the proteinencoded by the new DNA thus obtained in the same manner as above,whether or not the resulting DNA is the desired product can beconfirmed.

Examples of polynucleotides capable of hybridizing to the gene of thepresent invention are shown below. The M. furfur TIMM2782 strain usedherein has the MF-5 gene, as shown by SEQ ID NO:5, and also a genehaving the putative base sequence shown in FIG. 17, which has 90% ormore homology to the MF-5 gene base sequence. The proteins encoded bythe two genes each has homology to dihydrolipoamide dehydrogenase (DLDH)in the known protein. This strain also has the MF-6 gene as shown by SEQID NO:6, and also a gene having the putative base sequence as shown inFIG. 18, which has 90% or more homology to the MF-6 gene base sequence.The proteins encoded by the two genes each has homology to malatedehydrogenase (MDH) in the known protein. Moreover, the MF-1 gene (SEQID NO:1) and MF-2 gene (SEQ ID NO:2) of the present invention each has60% or more homology in terms of base sequence (FIG. 19) and aremutually capable of hybridizing. The proteins encoded by the two geneseach has homology to the peroxisome membrane protein PMP-20 from Candidaboidinii. Also, the MF-3 gene (SEQ ID NO:3) and MF-4 gene (SEQ ID NO:4)of the present invention each has 60% or more homology in terms of basesequence (FIG. 20) and are mutually capable of hybridizing. The proteinsencoded by the two genes each has homology to superoxide dismutase, andactually possess its enzyme activity. Accordingly, there are alsoencompassed in the present invention genes capable of hybridizing to thebase sequences of the present invention encoding the recombinantantigenic protein, the genes being possessed by other fungi being acausative of allergy.

The gene of the present invention is not particularly limited, and itmay be DNA or RNA, natural occurring or synthetic. Useful expressionvectors containing promoters, enhancers and other expression regulatoryelements suited for the expression of the gene of the present inventioninclude, for example, application of those described in “MolecularCloning, A Laboratory Manual, 2nd edition, J. Sambrook et al., published1989 by Cold Spring Harbor Laboratory.” Recombinant proteins expressedin mammalian, yeast, fungal or insect cells can undergo modifications,such as glycosylation and appropriate disulfide bonding. Availablevectors suitable for expression in yeast cells include pYES2, YepSec,and the like, which are made available. For those expressed in insectcells, the baculovirus vector is commercially available (manufactured byPharmingen, San Diego, Calif.), and for those expressed in mammaliancells, the pMSG vector is available (manufactured by Pharmacia).

In the case of those expressed in E. coli, the pTV118 vector, and thelike may be used. Also, when pMAL, pSEM, or pGEX is used, the gene ofthe present invention can be expressed as a fusion protein withmaltose-binding protein, with β-galactosidase, or with glutathioneS-transferase, respectively. In the case of those expressed as a fusionprotein, it is especially advantageous to introduce an enzymerecognition site at the location of the fusion joint between the carrierprotein and the antigenic protein from Malassezia or a fragment thereof.After isolating and purifying as a fusion protein, the desired antigenicprotein or fragment thereof can be selectively recovered by cleavage atthe enzyme recognition site and by subsequent biochemical purificationusing conventional methods. The enzyme recognition sites includerecognition sites of blood coagulation factor Xa or thrombin, andcommercial products may be used as these enzymes. It is also possible touse vectors capable of inducing expression by IPTG, temperature, or thelike.

Methods for introducing an expression vector into host cells are carriedout by conventional methods, such as the calcium phosphate or calciumchloride co-precipitation method, the DEAE-dextran method, or theelectroporation method.

(9) Antigenic Fragment of Present Invention

The present invention provides an antigenic fragment containing at leastone antigen epitope, and there are also included functional equivalentderivatives thereof. Specifically, the antigenic fragment of the presentinvention contains an antigen epitope contained in a recombinantMalassezia antigenic protein comprising an amino acid sequence as shownby any one of SEQ ID NOs:8 to 14 in Sequence Listing. The antigenicfragment of the present invention is characterized in that the antigenicfragment does not have a binding ability to IgE antibody specific toMalassezia fungi, or even when the antigenic fragment binds to the IgEantibody, such binding is at a level where no histamine is released frommast cells or basophiles. The antigenic fragment of the presentinvention is also characterized in that the antigenic fragment binds tothe IgE antibody at a substantially low level as compared to anantigenic protein from Malassezia. The antigenic fragment of the presentinvention is still also characterized in that the antigenic fragment hasa lower activity of activation of IgE-mediated immune response than thatof the antigenic protein.

The antigenic fragments of the present invention include antigenicfragments containing at least one T cell epitope. Alternatively, theremay be included antigenic fragments containing at least one B cellepitope, including, for instance, the antigenic fragments wherein theabove B cell epitope is selected from the amino acid sequences as shownby one of SEQ ID NOs:42 to 44 in Sequence Listing. These antigenicfragments may be chemically synthesized by means of peptide synthesistechniques, or they may be obtained as recombinant Malassezia allergensfrom host cells transformed a plasmid having a part of the gene andexpressing the desired epitope. For example, an antigenic protein may beprepared by optionally dividing the antigenic protein intonon-overlapping fragments of a desired length, preferably overlappingpeptide fragments of a desired length. The antigenicities of thesepeptide fragments are determined by assaying the binding of thesepeptide fragments to antibodies, or by assaying the effect on immuneresponse, including activation of T cell responses, induction of T cellanergy, and the like.

(10) Antibody or Fragments Thereof Against Recombinant MalasseziaAntigenic Protein of Present Invention or Antigenic Fragment Thereof

The present invention provides an antibody or fragments thereof whichspecifically binds to the above recombinant Malassezia antigenic proteinor antigenic fragments thereof. The antibody of the present inventioncan be obtained by a conventional method, and it may be polyclonalantibodies or monoclonal antibodies. The antibody fragment is notparticularly limited, as long as it specifically binds to the aboverecombinant Malassezia antigenic protein or fragments thereof.

(11) Synthetic Oligonucleotide Probe or Synthetic Oligonucleotide Primerof Present Invention

The present invention provides a synthetic oligonucleotide probe and asynthetic oligonucleotide primer capable of hybridizing to thepolynucleotide of the present invention. For example, there areencompassed in the present invention probes or primers containing anentire or partial sequence of the base sequences as shown by any one ofSEQ ID NOs:1 to 7. The gene encoding proteins having equivalentfunctions can be isolated by hybridization method using the probe. Thisprobe is prepared by, for instance, inserting the above gene orfragments thereof into an appropriate vector; introducing the vectorinto E. coli to replicate it; subsequently, extracting the replicatedproduct from the disrupted cell solution with phenol or the like;cleaving it with a restriction endonuclease that recognizes theinsertion site; carrying out electrophoresis, and cutting the desiredproduct from the gel. The probe can also be prepared on the basis of thebase sequences as shown by SEQ ID NOs:1 to 7 by chemical synthesis usingDNA synthesizers or by gene amplification technique using PCR. The aboveprobe may be labeled with a radioisotope or fluorescent substance toincrease its detection sensitivity upon use.

(12) Diagnostic Agent of Present Invention Containing as ActiveIngredient Recombinant Malassezia Antigenic Protein or AntigenicFragment Thereof

The present invention provides a diagnostic agent for Malasseziaallergoses or Malassezia infections, including, as an active ingredient,the recombinant Malassezia antigenic protein of the present invention orthe antigenic fragments thereof. The term “Malassezia allergoses” asused herein is defined as any allergoses of which causativemicroorganisms are Malassezia fungi, exemplified by atopic bronchialasthma, allergic rhinitis, allergic conjunctivitis, and atopicdermatitis. The term “Malassezia infections” is defined as anyinfectious disease of which causative microorganisms are Malasseziafungi, exemplified by tinea versicolor, Malassezia folliculitis, anddandruff.

The diagnostic agent for allergoses of the present invention is used asan intracutaneous diagnostic agent and titration reagent for allergydiagnosis in allergoses caused by Malassezia fungi. When used as anintracutaneous diagnostic agent, the recombinant antigenic protein ofthe present invention or the antigenic fragment of the present inventionis dissolved and diluted in phenol-containing physiological saline by aconventional method.

Also, when used as a titration reagent for allergy diagnosis, it can beprepared by a conventional method. For example, the recombinantantigenic protein of the present invention or the antigenic fragment ofthe present invention may be suitably dissolved and diluted in a Hanks'buffer to be used as a histamine release titration reagent. The methodcan be usually carried out by the following procedures. Specifically, agiven volume of blood of a patient with allergoses or a given number ofblood cells prepared by suspending a fraction of blood cells obtained bycentrifugation is titrated with a solution of the mentioned recombinantantigenic protein as a titration reagent by measuring the amount ofhistamine, which is released from basophiles, upon allergen stimulationby HPLC.

The recombinant antigenic protein of the present invention or theantigenic fragment of the present invention can also be used fordetection and diagnosis of Malassezia allergoses. For example, thediagnosis can be carried out by incubating blood or a blood componentsampled from a patient whose sensitivity to Malassezia fungi is toassessed, together with the isolated and purified, antigenic protein ofthe present invention, and the like under appropriate conditions, anddetermining the degree of binding of the purified, antigenic proteinwith a blood component, including, for instance, antibody, T cell, Bcell, or the like.

(13) Therapeutic Drug Containing Recombinant Malassezia AntigenicProtein or Antigenic Fragments of Present Invention as Active Ingredient

The present invention provides a therapeutic drug for Malasseziaallergoses or Malassezia infections including, as an active ingredient,the recombinant Malassezia antigenic protein or its antigenic fragmentsof the present invention. When the antigenic fragment from Malassezia isused for therapeutic purposes, it is preferred that the antigenicfragment binds to its IgE at concentrations substantially lower than thenaturally occurring Malassezia allergen, and that mediators are notreleased from mast cells or basophiles upon binding. More preferably,the antigenic fragment exhibits activity to activate T cell responseand/or is capable of inducing T cell anergy. A recombinant Malasseziaantigenic protein or antigenic fragments thereof can be assessed in invitro tests, such as RAST method, ELISA method, and histamine releasetests, as well as in skin tests and intracutaneous tests in laboratoryanimals or human volunteers.

The recombinant antigenic protein of the present invention and the genetherefor can be utilized for therapeutic drugs for Malasseziaallergoses. The therapeutic drug includes, as an active ingredient, theabove-described recombinant Malassezia antigenic protein, antigenicfragments thereof, or a peptide having an epitope, so that it can beutilized for therapeutic drugs for various allergoses caused byMalassezia fungi. Moreover, the above-described gene can also beutilized for a therapeutic drug, in which case the gene is inserted intoa vector expressible in a mammal and administered in the form of a DNAmolecule or viral particles having the gene in a suitable viral vector.By this administration, tolerance can be induced to treat diseases.

The method of preparing the therapeutic drug of the present inventionfor allergoses is not particularly limited. For example, the recombinantMalassezia antigenic protein prepared by the above method, or antigenicfragments thereof, or a peptide having an epitope, or a DNA moleculehaving a vector to which the above gene is inserted may be dried to apowder form and used as a hyposensitization therapeutic drug forallergoses caused by Malassezia fungi. When the therapeutic drug of thepresent invention for allergoses is used as a hyposensitizationtherapeutic drug, it can be used alone, or in the form of a combinationdrug containing commonly used adjuvants and various additives, such asstabilizers, excipients, dissolution aids, emulsifiers, buffers,soothing agents, preservatives, and coloring agents, which are added byconventional methods as occasion demands. For example, a purified,recombinant antigenic protein in the powder form is dissolved in aphenol-supplemented physiological saline and used for a stock solutionof an antigen for hyposensitization treatment.

The therapeutic drug of the present invention for allergoses can beadministered via ordinary pathways, including, for instance, oral,intracutaneous, subcutaneous, intramuscular, and intraperitonealpathways. Further, it can be used as percutaneous or transmucosal drugs,such as troches, sublingual tablets, eyedrops, intranasal sprays,poultices, creams, and lotions. Regarding the dosage and administrationfrequency of the therapeutic drug of the present invention forallergoses, the administration of the therapeutic drug can be suitablyselected so that the therapeutic drug is administered at a dose of aboutnot more than 20 mg per administration for an adult, depending onadministration pathways, symptoms, and the like, and about once everyweek. Also, the therapeutic drug of the present invention for allergosesis useful not only as a therapeutic drug but also as a prophylactic drugfor Malassezia allergoses. This is because it exhibits little or noanaphylaxis-inducing action and thus can be used safely in humans.

The therapeutic drug of the present invention for Malassezia allergosescontains as an active ingredient the above-described recombinant,antigenic protein or antigenic fragments thereof, and is used as atherapeutic drug and prophylactic drug for various Malasseziaallergoses. In order to use it as a hyposensitization therapeutic drug,it is particularly advantageous that the therapeutic agent has anepitope that does not bind to IgE specific to Malassezia fungi, or evenwhen the antigenic fragment binds to the IgE, the binding is at a levelwhere no histamine is released from mast cells or basophiles.

The present invention is hereinafter described in more detail by meansof the following working examples and comparative examples, withoutintending to limit the scope of the present invention thereto.

EXAMPLE 1 Isolation and Physicochemical Properties of Antigenic Proteinfrom Malassezia

1-1) Preparation of Malassezia Partially Purified Crude Antigen 2782

The culture was obtained by subjecting the M. furfur TIMM2782 strain(FERM BP-5611) to shaking culture at 27° C. for 5 days in fifty (50) 500ml conical flasks each containing 150 ml of Dixon medium (6.0% bactomalt extract broth, 2.0% Bacto Oxgall, 1.0% Tween 40, 0.25% glycerolα-monooleic acid). From the resulting culture, cells were harvested bycentrifugation. The cells were washed with a phosphate-buffered saline(PBS) five times, and the cells were then suspended in PBS in an amountdouble the wet weight of the cells, and disrupted and extracted byadding an equal amount of glass beads 0.5 mm in diameter, and using theMSK cell homogenizer (manufactured by B. Brown). The cell disruptionextract obtained was centrifuged (18,000 rpm, 30 min), and thesupernatant was obtained. The resulting supernatant was dialyzed againstpurified water and sterilized by filtration through a 0.45 μm membranefilter, followed by freeze-drying, to give about 900 mg of theMalassezia crude antigen 2782.

About 800 mg of the above Malassezia crude antigen 2782 was dissolved ina 0.05 M Tris-HCl buffer (pH 8.0) and subjected to ammonium sulfatesalting-out. The fraction precipitated on ammonium sulfate from 50% to90% saturation was collected by centrifugation, and the collectedfraction was dissolved in a 0.05 M Tris-HCl buffer (pH 8.0), and thesolution was subsequently dialyzed against the same buffer to give theMalassezia partially purified crude antigen 2782.

1-2) Screening for Antigenic Proteins from Malassezia

After freeze-drying, the Malassezia partially purified crude antigen2782 was dissolved in a 0.1 M potassium phosphate buffer (pH 7.0)containing 2 M ammonium sulfate so as to give a 4 mg/ml solution.Thereafter, 100 μl of the solution was applied to a column of PhenylSuperose PC 1.6/5 (column volume: 0.1 ml, manufactured by Pharmacia),previously equilibrated with the same buffer (pH 7.0) containing 2 Mammonium sulfate, and the elution was carried out with the same 0.1 Mbuffer on a linear gradient from 2 M to 0 M ammonium sulfate. Theantigenic protein-containing fraction obtained was dialyzed against aBis-Tris buffer (pH 6.5), and the dialyzed fraction was then applied toa column of Mono Q PC 1.6/5 (column volume: 0.1 ml, manufactured byPharmacia), and the elution was carried out with the same buffer on alinear gradient from 0 M to 0.3 M sodium chloride (FIG. 1, flow rate:100 μl/min, detection: 280 nm). The eluate was divided into 26 fractionsof 50 μl each, and the binding ability of IgE antibody was then examinedfor Fractions 1 through 20 by the Direct RAST (EIA) method using serafrom patients.

Specifically, each fraction was diluted 10 folds, 100 folds, and 1,000folds with a 0.1 M borate buffer (pH 8.0) containing 0.01% Tween 20, and45 μl of each dilution was coupled to a paper disc activated withcyanogen bromide and subsequently blocked with ethanolamine. Thereafter,each disc was supplemented with 50 μl of a 5-fold dilution of pooledsera (collection of sera from 10 patients showing high values in RASTmethod), followed by reaction with a diluted β-galactosidase-labeledgoat anti-human IgE antiserum. Thereafter, an enzyme substrate was addedthereto, followed by absorbance measurement at 415 nm. The results areshown in FIG. 2. It is clear from FIG. 2 that there are a plurality ofallergenic proteins. For example, a protein that binds to patient IgE ispresent in the neighborhoods of Fraction 6, and Fractions 12 and 13.

Separately, each fraction was subjected to SDS-PAGE, and it was stainedwith Coomassie Brilliant Blue (CBB) to detect proteins (FIG. 3), and therepresentative fractions were subjected to immunoblotting as describedbelow.

Specifically, each fraction was subjected to SDS-PAGE, and it was thentransferred onto a nitrocellulose membrane, blocked with 3% bovine serumalbumin (BSA), and treated with pooled sera from patients. Thereafter,the fraction was reacted with a diluted alkaline phosphatase-labeledgoat anti-human IgE antiserum, and an enzyme substrate was then added,followed by detection of allergenic protein. As a result, it is madeclear from FIG. 4 that there are a plurality of allergenic proteins. Forexample, it is evident that Fraction 12 contains a protein detected inthe neighborhood of 20 kDa on SDS-PAGE (isolated as an allergen MF-1),and the like, as allergenic proteins. It is also evident that Fraction 6contains an allergenic protein having a molecular weight of 20 kDa,nearly equal to that of Fraction 12 (isolated as an allergen MF-2), andanother protein detected in the neighborhood of 80 kDa, and the like.

1-3) Isolation of Purified Antigenic Proteins MF-1, MF-2, MF-3, MF-4,and MF-13

After 0.25 mg of a freeze-dried product of the above-describedMalassezia partially purified crude antigen 2782 was dissolved in 1 mlof a Bis-Tris buffer (pH 6.5) solution, the resulting solution wasapplied to a column of Mono Q HR 5/5 (column volume: 1 ml, manufacturedby Pharmacia) in the same manner as the Mono Q chromatography describedunder Item 1-2) above, resulting in four peaks, namely Peak 1(corresponding to Fractions 5 and 6 in FIG. 1), Peak 2 (corresponding toFractions 10, 11, and 12 in FIG. 1), Peak 3 (corresponding to Fractions15 and 16 in FIG. 1), and Peak 4 (corresponding to Fractions 18, 19, and20 in FIG. 1). Each peak was subjected to gel filtration chromatography,hydrophobic chromatography, and finally ion exchange chromatography byMono Q, to isolate pure antigenic proteins, wherein the protein namedMF-2 was isolated from Peak 1, that named MF-1 isolated from Peak 2,that named MF-3 isolated from Peak 3, and that named MF-4 isolated fromPeak 4. Separately, the Mono Q, non-adsorbed fraction of the Malasseziapartially purified antigen 2782 was subjected to hydrophobicchromatography to isolate a pure antigenic protein named MF-13. It wasconfirmed that the five isolated proteins were Malassezia allergenproteins by examining their binding ability of IgE antibody by EIAmethod using the above-described pooled sera from patients.

The purification method used is described in detail. Peaks 1 through 4as separated from Mono Q were each diluted 2 folds with a 0.1 Mpotassium phosphate buffer (pH 7.0) containing 4 M ammonium sulfate, andthereafter, the dilution was applied to a column of Phenyl Superose PC1.6/5 (column volume: 0.1 ml, manufactured by Pharmacia), previouslyequilibrated with a 0.1 M potassium phosphate buffer (pH 7.0) containing2 M ammonium sulfate, and the elution was carried out with the same 0.1M buffer on a linear gradient from 2 M to 0 M ammonium sulfate. Theantigenic protein-containing fraction obtained was concentrated using anultrafiltration membrane (MW 10,000), and the resulting concentrate wasthen subjected to gel filtration chromatography using the Sephadex G-75Superfine column (1.5×100 cm) to obtain a fraction eluted at a molecularweight of about 40,000. The gel filtration product obtained was furthersubjected to ion exchange chromatography using a column of Mono Q PC1.6/5, and elution was carried out in the same manner as above toisolate antigenic proteins. In other words, MF-1 was isolated from Peak2 (FIG. 5); MF-2 was isolated from Peak 1 (FIG. 6); MF-3 was isolatedfrom Peak 3 (FIG. 7); and MF-4 was isolated from Peak 4 (FIG. 8).Separately, the Mono Q non-adsorbed fraction was applied to the samecolumn of Phenyl Superose PC 1.6/5 (column volume: 0.1 ml, manufacturedby Pharmacia), and the elution was carried out with the same 0.1 Mbuffer on a linear gradient from 2 M to 0 M ammonium sulfate (FIG. 24)to isolate a pure, antigenic protein named MF-13.

1-4) Identification of MF-1 through MF-4 by Two-DimensionalElectrophoresis and Isolation of Purified, Antigenic Proteins MF-5Through MF-12

Further, 150 μg of the above-described Malassezia partially purifiedcrude antigen 2782 was dissolved in a solution containing 8 M urea, 0.5%NP-40, 2% β-mercapto-ethanol, 0.8% Pharmalyte (manufactured byPharmacia), and 0.01% Bromophenol Blue. First-dimensional isoelectricelectrophoresis was carried out by a conventional method using theImmobiline DryStrip gel (pH 4-7, manufactured by Pharmacia).Second-dimensional SDS-PAGE was carried out using the ExelGelSDS-Homogeneous (12.5%, manufactured by Pharmacia), followed by proteindetection by CBB staining (FIG. 9). After protein transfer onto a PVDFmembrane (manufactured by Millipore), immunoblotting was carried outusing sera from patients with allergoses (IgE antibodies) with apositive response to the crude antigen in skin test and a high value inRAST method, and normal individual sera (IgE antibodies) to detectpositive spots (FIG. 10). Of the positive spots found, those judged tohave high positive rate, namely, one having a molecular weight of about21 kDa and an isoelectric point of about 5.3; one having a molecularweight of about 20 kDa and an isoelectric point of about 5.8; one havinga molecular weight of about 27 kDa and an isoelectric point of about6.5; and one having a molecular weight of about 26 kDa and anisoelectric point of about 6.3 were identified as MF-1, MF-2, MF-3, andMF-4, respectively, based on the results of N-terminal sequencing, andthe like. Also detected were proteins having a molecular weight of about66 kDa and an isoelectric point of about 6.1 (named MF-5); a molecularweight of about 43 kDa and an isoelectric point of about 6.2 (namedMF-6); a molecular weight of about 15 kDa and an isoelectric point ofabout 6.0 (named MF-7); a molecular weight of about 30 kDa and anisoelectric point of about 5.4 (named MF-8); a molecular weight of about40 kDa and an isoelectric point of about 5.3 (named MF-9); a molecularweight of about 44 kDa and an isoelectric point of about 6.2 (namedMF-10); a molecular weight of about 45 kDa and an isoelectric point ofabout 6.4 (named MF-11); and a molecular weight of about 100 kDa and anisoelectric point of about 5.0 (named MF-12) as proteins binding to theIgE antibodies of the patients with allergoses. These proteins wereextracted from the gel and isolated.

1-5) Physicochemical Properties of Purified, Antigenic Proteins MF-1,MF-2, MF-3, MF-4, MF-5, MF-6, MF-7, MF-8, MF-9, MF-10, MF-11, MF-12, andMF-13

The isolated MF-1, MF-2, MF-3, MF-4, and MF-13 each showed a single bandin SDS-PAGE (FIG. 11). The results of analysis by SDS-PAGE andisoelectric electrophoresis of MF-1 through MF-13 are shown in Table 1.Isoelectric electrophoresis of MF-1 through MF-4 in non-denatured formwas carried out by a conventional method using IsoGel Plate at pH 3-10(manufactured by FMC). The results of analysis of SDS-PAGE andisoelectric electrophoresis of MF-5 through MF-12 were calculated fromthe results of two-dimensional electrophoresis shown in FIG. 9.

TABLE 1 SDS-PAGE (kDa) Under Reduced Under Non-Reduced IsoelectricConditions¹⁾ Conditions Point²⁾ MF-1 21 40 4.7 (5.3)  MF-2 20 40 4.8(5.8)  MF-3 27 27 5.2 (6.5)  MF-4 26 26 5.2 (6.3)  MF-5 66 — — (6.1)MF-6 43 — — (6.2) MF-7 15 — — (6.0) MF-8 30 — — (5.4) MF-9 40 — — (5.3)MF-10 44 — — (6.2) MF-11 45 — — (6.4) MF-12 100 — — (5.0) MF-13 16 — 8.1¹⁾Reduction: Treated with 3% of mercaptoethanol. ²⁾Numbers insidebrackets each indicate an isoelectric point in a denatured state with 8Murea.1-6) Preparation of Purified Antigenic Proteins MF-1, MF-2, MF-3, MF-4,and MF-13 in Large Amounts

A solution of the above-described Malassezia partially purified crudeantigen 2782 in a 0.05 M Tris-HCl buffer (pH 8.0) was adsorbed to acolumn of DEAE-cellulose, previously equilibrated with the same buffer.The column was washed with the same buffer followed by step-by-stepelution with the same buffer containing 0.1 M, 0.2 M, and 0.5 M sodiumchloride. The fraction eluted with the buffer containing 0.1 M sodiumchloride was concentrated using an ultrafiltration membrane (MW 10,000),and the concentrate was then subjected to column chromatography using acolumn of Sephacryl S-200HR (1.5×90 cm). The eluted fractions havingapparent molecular weights of 30,000 to 50,000 were collected andconcentrated using an ultrafiltration membrane (MW 10,000), and theconcentrates were then subjected to chromatography using the SephadexG-75 Superfine column (1.5×100 cm) to give Fraction 2 eluted at amolecular weight of about 40,000. This F2 fraction was dialyzed againsta 0.05 M Tris-HCl buffer (pH 8.0) containing 0.5 M sodium chloride, andthe dialyzed fraction was then subjected to chromatography using theChelating Sepharose Fast column (1×15 cm), previously chelated with zincions and equilibrated with the same buffer. The column was washed withthe same buffer followed by elution at buffers pH decreasing levels of7.0, 6.0, 5.0, and 4.0. The fraction eluted with the pH 5.0 buffer wascollected and concentrated, and the concentrate was then furtherpurified by chromatography using the Sephadex G-75 Superfine column(1.5×100 cm), to thereby isolate MF-2.

The effluent fraction in the zinc chelate chromatography wassubsequently purified by copper chelate chromatography. Specifically,the effluent fraction was subjected to chromatography using theChelating Sepharose Fast column (1×15 cm), previously chelated withcopper ions and equilibrated with a 0.05 M Tris-HCl buffer (pH 8.0)containing 0.5 M sodium chloride. The column was washed with the samebuffer, followed by elution at buffers of decreasing pH levels of 7.0,6.0, 5.0, and 4.0. The fraction eluted at pH 4.0 was concentrated usingan ultrafiltration membrane (MW 10,000), and the concentrate was thenfurther purified by chromatography using the above-mentioned SephadexG-75 Superfine column, to give MF-1 fraction eluted at a molecularweight of about 40,000. The resulting effluent fraction was concentratedusing an ultrafiltration membrane (MW 10,000), and the concentrate wasthen purified by chromatography using the above-mentioned Sephadex G-75Superfine column, to give a fraction eluted at a molecular weight ofabout 40,000. Thereafter, the eluted fraction was purified by anionexchange column chromatography of Mono Q, to isolate MF-3 and MF-4.

A portion of the above-described Malassezia partially purified antigen2782 fraction non-adsorbed to a DEAE-cellulose column was applied to acolumn of HiLoad 16/60 Superdex 75 pg (manufactured by Pharmacia),previously equilibrated with 0.05 M NH₄HCO₃, to collect a fractionhaving a molecular weight of not more than 20,000. The resultingfraction was adsorbed to HiTrap SP, previously equilibrated with a 0.05M acetate buffer (pH 5), and elution was carried out with the samebuffer supplemented with 0.2 M NaCl. The eluted fraction was applied toa column of HiLoad 16/60 Superdex 75 pg, previously equilibrated with0.05 M NH₄HCO₃, to isolate MF-13.

Finally, using about 0.5 g each of the Malassezia partially purifiedcrude antigen 2782 as a starting material, MF-1, MF-2, MF-3, MF-4, andMF-13 were obtained in amounts of 10 mg, 2 mg, 3 mg, 2 mg, and 2 mg,respectively. These antigenic proteins thus prepared in such largeamounts gave similar results as those described under Item 1-4) aboveand Example 10, in terms of SDS electrophoresis, isoelectricelectrophoresis, and N-terminal amino acid sequencing analysis.

EXAMPLE 2 Preparation of Monoclonal Antibodies

2-1) Mouse Immunization, Cell Fusion, and Hybridoma Cloning

Ten micrograms of each of the purified antigenic proteins MF-1, MF-2,and MF-3 as obtained in Example 1 was suspended in a Freund's completeadjuvant, and each suspension was intraperitoneally administered to maleBALB/c mice at 5 weeks of age. Four weeks later, 20 μg of an allergensuspended in a Freund's complete adjuvant was intraperitoneallyadministered for booster. Additional four weeks later, 20 μg of the sameallergen dissolved in a physiological saline was intravenouslyadministered.

Three days after final immunization, cell fusion was carried out bytaking out splenocytes and mixing with myeloma cells (P3X63-Ag8.653) ina 4:1 ratio, and then adding 43% polyethylene glycol 2000 thereto. Thismixture was sown into 96-well microplate wells at 2×10⁵splenocytes/well, and hybridomas were proliferated in an HAT mediumselectively. The presence of the desired antibody produced was examinedby ELISA using the culture supernatant to select antibody-producingcells. As a result, the 5B4 strain (FERM BP-5608) was obtained as aclone of a hybridoma that produces the M-40 monoclonal antibody againstthe purified antigenic protein MF-1; the 8G11 strain (FERM BP-5609) wasobtained as a clone of a hybridoma that produces the M-3 monoclonalantibody against the purified antigenic protein MF-2; and the 10C1strain (FERM BP-5610) was obtained as a clone of a hybridoma thatproduces the M-1 monoclonal antibody against the purified antigenicprotein MF-3.

2-2) Preparation of Ascites and Purification of Monoclonal Antibodies

To pristane-pretreated nude mice, 10⁷ hybridomas were intraperitoneallyinjected to allow hybridoma proliferation, and after one to two weeks,ascites was collected. From the resulting ascites, the monoclonalantibodies were purified using a protein A column kit (manufactured byAmersham), to give the M-40 monoclonal antibody against MF-1, the M-3monoclonal antibody against MF-2, and the M-1 monoclonal antibodyagainst MF-3. These monoclonal antibodies were all of the IgG1 isotype.

2-3) Preparation of Monoclonal Antibody-Immobilized Column andPurification of Antigenic Protein MF-3 Using Above Column

Fifteen milligrams of the above M-1 monoclonal antibody was dialyzedagainst a coupling buffer (0.1 M NaHCO₃, 0.5 M NaCl, pH 8.3), and thedialyzed monoclonal antibody was then coupled to 1 g of Sepharose 4B(manufactured by Pharmacia) activated with cyanogen bromide by aconventional method to prepare an antibody-immobilized resin.

The resin obtained was transferred into a lesser column of 5 mlcapacity. A solution of 40 mg of the Malassezia partially purified crudeantigen 2782 in a 0.05 M Tris-HCl buffer (pH 8.0) was applied to thecolumn. After the column was thoroughly washed with a 0.1 M Tris-HClbuffer (pH 8.0), elution of the antibody-bound antigenic protein wascarried out with a 0.1 M glycine-HCl buffer (pH 2.5). The eluate wasimmediately made neutral again by the addition of a 1 M Tris-HCl buffer(pH 8.0), and the neutralized eluate was then concentrated using anultrafiltration membrane (MW 10,000), followed by gel filtrationchromatography using the Sephadex G-75 Superfine column (1.5×100 cm) inthe same manner as above, to isolate about 300 μg of MF-3 of highpurity.

EXAMPLE 3 Diagnostic Application of Purified Antigenic Proteins

3-1) Determination of Specific IgE Antibodies by RAST Method

Paper disc activation with cyanogen bromide and coupling of purifiedallergens to the paper disc were carried out according to the method ofMiyamoto et al. (Allergy, 22, 584-594, 1973). One paper disc coupledwith the allergen and 50 μl of sera from patients were added to apolystyrene tube, followed by incubation at room temperature for 3hours. The paper disc was washed three times with a physiological salinecontaining 0.2% Tween 20, and 50 μl of the ¹²⁵I-labeled anti-human IgEantibody of the RAST-RIA kit, manufactured by Pharmacia, was then added,followed by overnight incubation at room temperature. The disc waswashed three times again, and radioactivity was then determined using agamma counter. From a standard curve prepared from a simultaneousradioactivity determination with a reference reagent of the kit, the IgEantibody titer was calculated. For samples that yielded values exceedingthe upper limit of the standard curve (>17.5 PRU/ml), the antibody titerwas calculated after the samples were diluted 10 folds or 100 folds inequine sera and assayed again.

3-2) Diagnosis Using Purified, Antigenic Proteins MF-1, MF-2, MF-4, andMF-13

A skin test using a Malassezia crude antigen was carried out on patientswith atopic dermatitis (hereinafter abbreviated AD) or bronchial asthma(hereinafter abbreviated BA) or both (AD+BA). Positive response wasobserved in 43 out of 57 AD patients (75%), 108 out of 919 of BApatients (12%), and 47 out of 102 AD+BA patients, demonstrating anextremely high positivity rate in the AD patients. Also, 100%, 59%, and85%, respectively among these AD, BA, and AD+BA patients with positiveskin tests, were positive in IgE antibody determination by RAST method.

On the 76 patients (AD patients: 30, BA patients: 20, AD+BA patients:26) positive both in the skin test using the Malassezia crude antigenand in RAST method (1 or higher score), IgE antibody titers againstthree purified antigenic proteins, i.e., MF-1, MF-2, and MF-4, weredetermined by RAST method (RIA method). IgE antibody titers forantigenic proteins were determined on 12 normal individuals withnegative skin tests as well in the same manner as above. As a result, itwas made clear from Table 2 that IgE antibodies against the antigenicproteins were present in sera from patients at very high rates.Especially high positivity rates were obtained against MF-1 and MF-2.Further, there were patients with surprisingly very high IgE antibodytiters (Table 3), and particularly the mean titer against MF-1 and MF-2for the AD patients was 100 PRU, and there were some patients withhighest values exceeding 1,000 PRU. Also, the sera from all patientspositive to the Malassezia crude antigen in RAST method contained theIgE antibody against any one of the purified antigenic proteins MF-1,MF-2, and MF-4.

Also the IgE antibody titer against MF-13 by RAST method for 11 ADpatients positive both in the skin test using the Malassezia crudeantigen and in RAST method. As a result, nine out of 11 patients werefound to be positive in RAST.

TABLE 2 Patients with Allergoses (Rate of RAST Positive) normal AD + BAAD Total individuals BA (n = 20) (n = 26) (n = 30) (n = 76) (n = 12)MF-1 100 (20/20) 96 (25/26) 90 (27/30) 95 (72/76) 0 (0/12) MF-2 100(20/20) 100 (26/26)  87 (26/30) 95 (72/76) 0 (0/12) MF-4  75 (15/20) 88(23/26) 87 (26/30) 84 (64/76) 0 (0/12) BA: Patients with allergicasthmatics. AD: Patients with atopic dermatitis. AD + BA: Patients withAtopic dermatitis and allergic asthmatics complications.

TABLE 3 Patients with Allergoses [IgE Antibody Titer (PRU Value)] normalIndividuals BA (n = 20) AD + BA (n = 26) AD (n = 30) (n = 12) MF-1 1.65± 0.66 14.73 ± 4.15 119.73 ± 56.95 <0.35 MF-2 4.32 ± 2.59 16.01 ± 4.45112.84 ± 52.23 <0.35 MF-4 3.54 ± 2.08  9.75 ± 2.43  94.75 ± 42.43 <0.35BA: Patients with allergic asthmatics. AD: Patients with atopicdermatitis. AD + BA: Patients with Atopic dermatitis and allergicasthmatics complications.3-3) Immunological Properties of Purified Antigenic Proteins MF-1, MF-2,MF-3, and MF-4

A RAST cross inhibition test using pooled sera from patients was carriedout to evaluate cross reactivity among three purified antigenic proteins(MF-1, MF-2, MF-4) (Table 4). As a result, it was shown that they didnot mutually cause cross-reactivity, namely that the specific IgEantibodies against the respective purified antigenic proteins arepresent in the sera from patients.

TABLE 4 Antigen Concentration of Various Antigens Required forImmobilized 50% Inhibition of Binding Antigen Immobilized on Solid onSolid Phase and Patient IgE (μg/ml) Phase MF-1 MF-2 MF-4 MF-1 0.038(1)    8.6 (230)   52 (1370) MF-2 >100 (>7700) 0.013 (1)    >100 (>7700)MF-4 18 (290) 30 (480) 0.062 (1)   

Next, the purified antigenic proteins MF-1, MF-2, and MF-4 were stepwisediluted and their antigen potencies were determined by the Direct RASTEIA method. Specifically, dilutions of the purified, antigenic proteinMF-1, MF-2, and MF-4 were each coupled to a cyanogen bromide-activatedpaper disc and then the coupled purified, antigenic protein was blockedwith ethanolamine. Thereafter, 50 μl of a 5-fold dilution of pooled serawas then added to each disc, and the mixture was reacted with a dilutedβ-galactosidase-labeled goat anti-human IgE antiserum. Thereafter, anenzyme substrate was added, followed by absorption determination at 415nm. The results are shown in FIG. 12. It is clear that MF-1 binds tosera from patients IgE at the lowest concentration.

Separately, the purified antigenic protein MF-3 was stepwise diluted,and its antigen potency was determined by ELISA. Specifically, afterapplying each dilution of the purified antigenic protein MF-3 to amicroplate, the microplate was washed with a physiological salinecontaining 0.01% Tween 20, blocked with PBS containing 3% BSA, washedwith a physiological saline containing 0.01% Tween 20, and then pooledsera were added. The microplate was kept standing at 37° C. for 2 hours,and a secondary antibody, a peroxidase-labeled goat anti-human IgEantiserum was added, and subsequently a substrate solution was added;after color development, absorbance at 450 nm was determined. Theresults are shown in FIG. 13.

EXAMPLE 4 Preparation of Pyridylethylated Derivative of Cysteine Residueof Purified, Antigenic Protein MF-2

The purified antigenic protein MF-2 (0.04 mg) was dissolved in 200 μl ofa borate-buffered saline (pH 8.0). To this solution were added 800 μl of5 M guanidine hydrochloride, 1 μl of 4-vinylpyridine, and 2 μl oftributyl phosphine. After replacing the atmosphere with nitrogen gas,reaction was carried out overnight at 37° C., and the resulting mixturewas subjected to isolation and purification by HPLC (column:μ-Bondasphere C4-300, 2×150 mm, manufactured by Waters; solvents:washing with 0.05% TFA/water for 15 minutes, followed by linear gradientelution so as to give 80% acetonitrile containing 0.05% TFA after 60minutes; flow rate: 220 μl/min.; detection: 220 nm; column temp.: 40°C.; FIG. 14). The product obtained was identified as thepyridylethylated product of MF-2, from the fact that its band appearedin the neighborhood of 20 kDa in SDS electrophoresis under non-reducedconditions (in absence of mercaptoethanol), and that the peptidefragments (FIG. 15) which have the N-terminal amino acid sequences asshown by SEQ ID NOs:47 and 48 (eluted at 28.20 and 31.15, respectively),obtained by lysylendopeptidase digestion of the product obtained had apyridylethylcysteine group. The pyridylethylated MF-2 obtained, whichwas similar to MF-2, was confirmed to be bound to sera IgE of patientsfrom Malassezia allergoses by immunoblotting after SDS electrophoresis.

EXAMPLE 5 Isolation of Antigenic Fragment Peptide Derived from PurifiedAntigenic Protein MF-3

The purified antigenic protein MF-3 (0.04 mg) was dissolved in 100 μl ofa borate-buffered saline (pH 8.0). To this solution were added 900 μl of5 M guanidine hydrochloride, 1 μl of 4-vinylpyridine, and 2 μl oftributyl phosphine. After replacing the atmosphere with nitrogen gas,reaction was carried out overnight at 37° C., and the resulting mixturewas subjected to isolation and purification by HPLC (column:μ-Bondasphere C4-300, 2×150 mm, manufactured by Waters; solvents:washing with 0.05% TFA/water for 15 minutes, followed by linear gradientelution so as to give 80% acetonitrile containing 0.05% TFA after 60minutes). To the resulting purified, antigenic protein MF-3 treated withguanidine hydrochloride were added, 100 μl of 50 mMN-ethylmorphine-acetic acid (pH 9.0) and lysylendopeptidase(Achromobacter protease I, manufactured by Wako Pure ChemicalIndustries), followed by reaction carried out overnight at 37° C.Thereafter, the reaction mixture was subjected to HPLC (column:μ-Bondasphere C18-300, 2×150 mm, manufactured by Waters; solvents:linear gradient elution from 0.05% TFA/water eluted so as to give 60%acetonitrile containing 0.05% TFA; flow rate: 200 μl/min.; detection:214 nm; column temp.: 40° C.; FIG. 16). Each peptide fragment wasseparately collected and freeze-dried, and thereafter the freeze-driedfragment was assayed for binding to sera IgE of patients from Malasseziaallergoses by ELISA as described below.

Specifically, each peptide fragment (about 10 to 100 pmol for each) wasspread onto a microplate using a peptide coating kit (manufactured byTakara Shuzo Co., Ltd.) and then washed with a physiological salinecontaining 0.01% Tween 20. The washed microplate was blocked with 3%BSA, and treated with the sera from patients. Thereafter, each peptidefragment was then reacted with a diluted peroxidase-labeled goatanti-human IgE antibody, and an enzyme substrate was added thereto.After a given period of time, absorbance was determined to detectantigenic fragments. As a result, there appeared to show the antigenicfragments that were bound to patient serum IgE were present in peakseluted around 20.02, 21.41, and 24.07 minutes. Of these peaks, the21.41-minute peak was found to contain a peptide having an amino acidsequence consisting of HHQTYVNNLNAAXK (SEQ ID NO:58, wherein X is anundetermined amino acid).

EXAMPLE 6 Lymphocyte Blast Formation Test

Heparinized venous blood samples were collected from subjects [eightpatients with allergoses (Nos. 1 through 8 in Table 5), two normalindividuals (Nos. 9 and 10 in Table 5)], and lymphocytes were separatedby the Ficoll gravitational centrifugation method. After preparationwith a 10% FCS-supplemented RPMI1640 medium so as to give a cell numberof 5×10⁵ cells/ml, this suspension was poured onto 96-well microplatesat 0.2 ml per plate. The above Malassezia partially purified crudeantigen 2782 was added so as to have concentrations of 10 and 100 μg/ml,and the purified, antigenic proteins (MF-1, MF-2, and MF-4) were eachadded so as to have concentrations of 1 and 10 μg/ml, followed by fivedays of cultivation in the presence of 5% CO₂ at 37° C. underhigh-humidity conditions. In the forth day, 0.5 μCi tritiated(³H)-thymidine was added. After completion of the cultivation,lymphocytes were harvested and assayed for the amount of ³H-thymidineuptake using a liquid scintillation counter. Using the mean value forthree runs, the ratio of the amount of the ³H-thymidine uptake of theantigen-added and non-added groups was expressed as the SI (stimulationindex). The results are shown in Table 5. It is clear from Table 5 thatthe lymphocytes derived from Patient No. 4 proliferated in response tothe purified, antigenic proteins MF-1 and MF-2, and that those derivedfrom Patient Nos. 1 and 6 proliferated especially in response to MF-2.

TABLE 5 SI (in case of adding low allergen concentration/in case ofadding high allergen concentration)*⁾ 1 2 3 4 5 6 7 8 9 10 MF-1 7.7/2.54.3/1.4 1.0/0.9 4.2/3.7 2.6/2.0 2.1/1.0 1.7/1.2 2.1/1.7 1.1/0.5 2.0/0.7MF-2 4.0/2.9 1.3/1.5 1.9/1.2 7.8/4.2 2.3/2.3 3.1/2.6 2.0/1.8 1.4/1.72.0/0.7 1.6/1.0 MF-4 1.8/1.3 1.2/1.1 1.0/0.9 2.5/1.4 1.2/1.8 1.9/1.71.1/0.9 1.3/1.3 1.9/0.8 0.9/0.6 Remarks *⁾: “In case of adding lowallergen concentration” refers to a case of adding 1 μg/ml MF-1, MF-2,or MF-4. “In case of adding high allergen concentration” refers to acase of adding 10 μg/ml MF-1, MF-2, or MF-4. 1-8: Allergic patients.9-10: Normal individuals.

EXAMPLE 7 Preparation of Diagnostic Reagent for Intracutaneous Reactionand Preparation of Titration Reagent for Diagnosis Against MalasseziaAllergy

A purified allergen-active component is dried and collected in a powderform to be used as a diagnostic reagent for intracutaneous reactionagainst Malassezia allergoses and as a titration reagent for thediagnosis of the Malassezia allergy. The diagnostic reagent forintracutaneous reaction is prepared by 200,000-fold dilution of theallergen-active component using a 0.9% physiological saline containing0.5% phenol as a solvent. The titration reagent for the diagnosis of theMalassezia allergy is prepared by dissolving the allergen-activecomponent in a Hanks' buffer at a concentration of 1 mg/ml, to give astock solution for a titration reagent for histamine release, using thedilutions of the stock solution.

EXAMPLE 8 Preparation of Antigenic Agent for Hyposensitization Therapy

A purified allergen-active component is dried and collected in a powderform to be used as a hyposensitization therapeutic agent for Malasseziaallergoses. The allergen-active component is dissolved in a 0.9% salinecontaining 0.5% phenol at a concentration of 1 mg/ml to give a stocksolution of an antigen for hyposensitization therapy.

EXAMPLE 9 Quantitative Assay of Purified, Antigenic Protein MF-1 inHouse Dust and Cultivation of Malassezia

House dust was collected from rooms, bedclothes, and the like, in housesinhabited by bronchial asthma patients, using a vacuum cleaner undergiven conditions. MF-1 was subjected to quantitative assay by means ofsandwich ELISA using a rabbit polyclonal antibody and the mousemonoclonal antibody (M-40) as obtained in Example 2-2), and asupernatant obtained from 1:10 (w/v) extraction of the dust was used asa sample for quantitative assay of MF-1. In order to cultivateMalassezia, the dust was suspended in sterile water in a 1:10 (w/v)ratio and sown over a plate medium. Also, a sterile tape was onceattached to the bedclothes surface, removed, and placed on the platemedium. The media used were PDA, M40YA, or a Dixon agar medium, and thenumber of colonies was counted after cultivation at 25° C. for one week.

It is possible to subject MF-1 to quantitative assay of the level of notless than 1 ng/g dust by sandwich ELISA method, by which 87.1 to 1.1ng/g dust of MF-1 was detected in 16 out of 24 dust samples derived frombedclothes. As for the cultivation results for Malassezia on thebedclothes surface, obtained by the tape method, 10 out of the 24samples were positive. Incidentally, out of the 24 samples, 14 samples(58%, eight being positive, six being negative) gave results inagreement with those of MF-1 detection by sandwich ELISA method andcultivation.

EXAMPLE 10 Determination of Partial Amino Acid Sequences of Purified,Antigenic Proteins MF-1, MF-2, MF-3, MF-4, MF-5, MF-6, MF-7, MF-10, andMF-13

N-terminal amino acid sequence analysis was carried out by aconventional method. As a result, it was made clear that MF-1 has theamino acid sequence:

(SEQ ID NO:45) Pro Gly Asp Pro Thr Ala Thr Ala Lys Gly Asn Glu Ile ProAsp Thr Leu Met Gly Tyr Ile Pro Trp Thr Pro Glu Leu Asp

As for MF-2, since its N-terminal is blocked, pyridylethylation wasfollowed by lysylendopeptidase digestion. The resulting peptidefragments were analyzed by C18 reversed-phase HPLC. The various peaksobtained were separately collected, some of which were subjected toamino acid sequencing determination. The three peptide fragments elutedat 27.07 minutes, 28.20 minutes, and 31.15 minutes, respectively, weredetermined to have the following respective N-terminal amino acidsequences:

(SEQ ID NO:46) Val Glu Tyr Phe Gly Ile Asp Glu Gly Glu Pro Lys; (SEQ IDNO:47) Asp Asn Leu Thr Phe Ala Gln Asp Val Asn Cys Glu Phe; (SEQ IDNO:48) Val Val Ile Val Ala Val Pro Gly Xaa Phe Thr Pro Thr Cys Thr AlaAsn His Val Pro Xaa Tyr Xaa Glu

wherein Xaa is an undetermined amino acid.

As for MF-3, since its N-terminal is also blocked, pyridylethylation wasfollowed by lysylendopeptidase digestion. The resulting peptidefragments were analyzed by C18 reversed-phase HPLC. The various peaksobtained were separately collected, some of which were subjected toamino acid sequencing determination. The three peptide fragments elutedat 35.68 minutes, 36.68 minutes, and 29.15 minutes, respectively, weredetermined to have the following respective N-terminal amino acidsequences:

(SEQ ID NO:49) Asp Gln Asp Pro Leu Thr Thr His His Pro Val Ile Gly TrpAsp Xaa Xaa Glu His Ala

wherein Xaa is an undetermined amino acid;

(SEQ ID NO:50); Ala Trp Trp Asn Val Val Asn Trp Ala Glu Ala Glu Lys (SEQID NO:51) Phe Xaa Gly Gly Gly His Ile Asn Xaa Ser Leu Phe

wherein Xaa is an undetermined amino acid.

In addition, as a result of N-terminal amino acid sequence analysis, itwas made clear that MF-4 had the amino acid sequence:

(SEQ ID NO:52) Lys Tyr Thr Leu Pro Pro Leu Pro Tyr Asp Tyr Gly Ala LeuGlu Pro Ala Ile Ser Gly Glu Ile Met Glu Thr His Tyr Glu Lys His

In addition, as a result of N-terminal amino acid sequence analysis, itwas made clear that MF-5 had the amino acid sequence:

(SEQ ID NO:53) Xaa Xaa Xaa Xaa Xaa Glu Pro Tyr Asp Val Ile Val Ile GlyGly Gly Pro Gly Gly Tyr Val Ala Xaa Xaa Lys Xaa Xaa Gln

wherein Xaa is an undetermined amino acid.

In addition, as a result of N-terminal amino acid sequence analysis, itwas made clear that MF-6 had the amino acid sequence:

(SEQ ID NO:54) Arg Lys Val Ala Val Leu Gly Ala Ser Gly Gly Ile Gly GlnPro Leu Ser Leu Leu Met Lys Leu Asn Pro Lys Val Thr Glu Leu Arg

In addition, as a result of N-terminal amino acid sequence analysis, itwas made clear that MF-7 had the amino acid sequence:

(SEQ ID NO:55) Gly Asn Asn Gly Leu Ser Glu Val Val Tyr Lys Pro Asp XaaGln Xaa Thr Xaa Glu Phe Xaa Val Ile

wherein Xaa is an undetermined amino acid.

In addition, as a result of N-terminal amino acid sequence analysis, itwas made clear that MF-10 had the amino acid sequence:

Val Asp Gln Xaa Tyr Phe Gly Leu Xaa (SEQ ID NO:56)

wherein Xaa is an undetermined amino acid.

In addition, as a result of N-terminal amino acid sequence analysis, itwas made clear that MF-13 had the amino acid sequence:

(SEQ ID NO:57) Ser Asn Val Phe Phe Asp Ile Thr Lys Asn Gly Ser Pro LeuGly Thr Ile Lys Phe Lys Leu Phe Asp Asp Val

The other antigenic proteins could not be analyzed due to N-terminalblocking, and the like.

As a result of homology searching with known proteins, it was made clearthat MF-2 is a protein having the partial amino acid sequence of SEQ IDNO:48 homologous to a peroxisome membrane protein (PMP-20) derived fromCandida boidinii, and MF-3 is a protein having the above partial aminoacid sequence homologous to iron/manganese-superoxide dismutase. Inaddition, it was made clear that MF-4 is a protein having the aboveN-terminal amino acid sequence homologous to iron/manganese-superoxidedismutase in the same manner as in MF-3. In addition, it was made clearthat MF-5 is a protein having the above N-terminal amino acid sequencehomologous to dehydeolipoamide dehydrogenase. In addition, it was madeclear that MF-6 is a protein having the above N-terminal amino acidsequence homologous to malate dehydrogenase. In addition, as for MF-7and MF-10, no homology to known proteins was found from their N-terminalamino acid sequences. In addition, it was made clear that MF-13 is aprotein having the above N-terminal amino acid sequence homologous tocyclophilin.

EXAMPLE 11 Cloning of Antigenic Protein MF-1 Gene from M. furfur

11-a) Purification of Total RNA from M. furfur

In order to obtain total RNA from cells of the M. furfur TIMM2782strain, the strain was cultured for 72 hours in 300 ml of a YNB medium(0.67% bacto yeast nitrogen DNA, 0.5% Bacto Casiton, 0.1% Tween 60, 2.0%glucose, 5% MEM-vitamin solution), and the cells were then harvested bycentrifugation at 3,000 rpm for 15 minutes. The harvested cells wererapidly frozen with liquid nitrogen. The frozen cells were disruptedinto a powder form by a mortar, and 1.3 mg of the total RNA was thenrecovered and purified by an RNA extraction kit (manufactured byPharmacia).

11-b) Amplification of MF-1 Gene by RT-PCR

The oligonucleotides MF1F1 and MF1F2, deduced from the amino acidsequence for the N-terminal of the MF-1 protein described in Example 10were synthesized and purified to be used as primers for PCR. The basesequences for MF1F1 and MF1F2 are shown by SEQ ID NOs:15 and 16,respectively, in Sequence Listing. An MF-1 cDNA was amplified by RT-PCRusing RNA PCR Kit Ver. 2 (manufactured by Takara Shuzo Co., Ltd.) with 1μg of the total RNA purified in Example 11-a). Specifically, the cDNAwas synthesized from 1 μg of the total RNA by an AMV reversetranscriptase reaction (at 42° C. for 60 minutes) using anoligo(dT)₂₀-M4 adaptor primer. PCR reaction was carried out by repeating40 cycles of the temperature shifts at 94° C. for 1 minute, at 55° C.for 2 minutes, and at 72° C. for 1.5 minutes, using the MF1F1 primer andthe M13M4 primer included in the kit together with this cDNA as atemplate. Second PCR reaction (nested PCR reaction) was carried outusing this PCR reaction mixture as a template. The MF1F2 primer and theM13M4 primer were used in this reaction. As a result of the PCR, a cDNAfragment with about 570 bp in length was amplified. This cDNA was clonedinto a pUC118 vector (manufactured by Takara Shuzo Co., Ltd.), and itsbase sequence was then determined. The resulting base sequence is shownby SEQ ID NO:17 in Sequence Listing. The amino acid sequence deducedfrom SEQ ID NO:17 was identical to the amino acid sequence determinedfrom the MF-1 protein. Therefore, it is clearly demonstrated that thiscDNA fragment is an MF-1 gene.

11-c) Preparation of M. furfur cDNA Library

20 μg of poly(A)⁺ RNA was purified from 1 mg of the total RNA obtainedin Example 11-a) with Oligotex-dT30<SUPER> (manufactured by Takara ShuzoCo., Ltd.). A cDNA was synthesized by a cDNA synthesis kit (manufacturedby Takara Shuzo Co., Ltd.) using 5 μg of the poly(A)⁺ RNA. A cDNAlibrary was constructed by ligating the synthesized cDNA and the lambdaphage vector λSH1ox™ (manufactured by Novagen) together, and carryingout in vitro packaging using Phagemaker System and Phage Pack Extract(manufactured by Novagen).

11-d) Cloning of MF-1 cDNA

The cDNA library obtained in Example 11-c) was infected into a hostEscherichia coli ER1647 strain and mixed with Top Agarose (an LB mediumcontaining 0.7% bacto agar), and a plaque was then formed by overlayingon an LB plate and culturing at 37° C. overnight. The resulting plaquewas transferred onto a nylon membrane (“Hybond-N,” manufactured byAmersham) and subjected to plaque hybridization. A cDNA fragment of MF-1with about 570 bp obtained in Example 11-b) was labeled with [α-³²P]dCTPusing a random primer DNA labeling kit (manufactured by Takara ShuzoCo., Ltd.), and the labeled cDNA fragment was used as a probe forhybridization. 1.6×10⁵ plaques were screened for, and 10 clones withstrong signals out of the positive clones were then subjected to furtheranalysis. Specifically, E. coli cells harbouring the plasmid which has aregion containing the MF-1 cDNA were obtained from these phages byautomatic subcloning in E. coli. The plasmids were purified from theseE. Coli cells, and pMF1-7, which contained the longest fragment withabout 600 bp cDNA, was selected. The cDNA was subcloned into a pUC118vector (manufactured by Takara Shuzo Co., Ltd.), and its base sequencewas then determined. The base sequence thereof is shown by SEQ ID NO:1in Sequence Listing, and the MF-1 gene encodes a polypeptide having anamino acid sequence as shown by SEQ ID NO:8 in Sequence Listing.

11-e) Purification of Genomic DNA from M. furfur

In order to obtain a genomic DNA from cells of the M. furfur TIMM2782strain, the strain was cultured for 72 hours in 200 ml of the YNBmedium, and the cells were harvested by centrifugation at 3,000 rpm for15 minutes. The harvested cells were washed with a washing solution(0.9% NaCl, 0.05% Tween 80) five times, and then with a PK buffer (0.15M NaCl, 0.1 M Tris-HCl (pH 7.5), 10 mM EDTA) three times. The cells weresuspended in 8 ml of the PK buffer, and an equivolume of glass beads(425 to 600 μm in diameter, manufactured by Sigma) was then addedthereto, and the cells were disrupted using mini-bead beater(manufactured by Biospace). Protease K and SDS were added to the celldisruption, so as to have final concentrations of 0.15 mg/ml and 1%(w/v), respectively, and the resulting mixture was treated at 50° C. for3 hours while gently stirring the mixture. The nucleic acid was purifiedby subjecting the disrupted solution to phenol extraction,phenol/chloroform extraction, and chloroform extraction (each carriedout once), and subjected to ethanol precipitation. The nucleic acidobtained by centrifugation at 10,000 rpm for 15 minutes was dissolved ina TE buffer (10 mM Tris-HCl, 1 mM EDTA). RNase A was added to thenucleic acid solution so as to have a final concentration of 40 μg/ml,and the mixture was treated at 37° C. for 40 minutes. The DNA wasrecovered and purified by subjecting the solution to phenol extraction,phenol/chloroform extraction, and chloroform extraction (each carriedout once), and by subjecting to ethanol precipitation.

11-f) Cloning of MF-1 Genomic DNA

The genomic DNA obtained in Example 11-e) was completely cleaved withBamHI or PstI, and each of the resulting fragments was then cloned intothe pUC118 vector to prepare two kinds of genomic DNA libraries. An MF-1genomic DNA was screened from the libraries by colony hybridizationusing the MF-1 cDNA obtained in Example 11-d) as a probe. A clonecontaining an 8.5 kbp DNA was obtained from the library containing aBamHI fragment, and a clone containing a 4.9 kbp DNA was obtained fromthe library containing a PstI fragment. Based on the base sequence ofthe cDNA, the base sequence of the 4.9 kbp PstI fragment was determined.The base sequence of the genomic DNA containing the MF-1 gene is shownby SEQ ID NO:18 in Sequence Listing. According to this base sequence,the MF-1 gene encodes a polypeptide having an amino acid sequence asshown by SEQ ID NO:19 in Sequence Listing.

Further, it is made clear that there are two introns each with 37 bp and39 bp in the genomic DNA. The relationship between the genomic DNA andthe cDNA is shown in FIG. 23.

EXAMPLE 12 Cloning of Antigenic Protein MF-2 Gene from M. furfur

12-a) Amplification of MF-2 Gene by RT-PCR

The oligonucleotide MF2F1 deduced from the internal amino acid sequenceof the MF-2 protein described in Example 10 was synthesized and purifiedto be used as a primer for PCR. The base sequence of MF2F1 is shown bySEQ ID NO:20 in Sequence Listing. An MF-2 cDNA fragment was amplified bycarrying out RT-PCR according to the method described in Example 11-b)using the MF2F1 and M13M4 primers. As a result of the first PCRreaction, a cDNA fragment with about 280 bp in length was amplified. Thebase sequence of the cDNA fragment amplified is shown by SEQ ID NO:21 inSequence Listing. The amino acid sequence deduced from SEQ ID NO:21 wasidentical to the amino acid sequence determined from the MF-2 protein.Therefore, it is clearly demonstrated that this cDNA fragment is an MF-2gene.

12-b) Cloning of MF-2 cDNA

Plaque hybridization was carried out using the MF-2 cDNA fragment withabout 280 bp as shown by SEQ ID NO:21 obtained in Example 12-a) as aprobe according to the method described in Example 11-d). Ten cloneswith strong signals out of positive clones were subjected to furtheranalysis. Specifically, E. coli cells harbouring the plasmid which has aregion containing an MF-2 cDNA were obtained from these phages byautomatic subcloning in E. coli. The plasmids were purified from theseE. coli cells, and pMF2-2, which contained the longest fragment withabout 550 bp cDNA, was selected. The cDNA was subcloned into a pUC118vector, and its base sequence was then determined. The base sequence isshown by SEQ ID NO:2 in Sequence Listing, and the MF-2 gene encodes apolypeptide having an amino acid sequence as shown by SEQ ID NO:9 inSequence Listing.

EXAMPLE 13 Cloning of Antigenic Protein MF-3 Gene from M. furfur

13-a) Amplification of MF-3 Gene by RT-PCR

The oligonucleotides MF3F1, MF3F2, and MF3F3 deduced from the internalamino acid sequence of the MF-3 protein described in Example 10 weresynthesized and purified to be used as primers for PCR. The basesequences of MF3F1, MF3F2, and MF3F3 are shown by SEQ ID NOs:22 to 24 inSequence Listing, respectively. An MF-3 cDNA fragment was amplified bycarrying out RT-PCR according to the method described in Example 11-b)using MF3F1 and M13M4 primers in the first PCR reaction, and using acombination of MF3F1 and MF3R3 primers and a combination of MF3F2 andM13M4 primers in the second PCR reaction. As a result of the PCRreaction, a cDNA fragment with about 380 bp in length was amplified forthe combination of MF3F1 and MF3R3 primers, and a cDNA fragment withabout 280 bp in length was amplified for the combination of MF3F2 andM13M4 primers. The base sequences of the cDNA fragment amplified areshown by SEQ ID NOs:25 and 26 in Sequence Listing, respectively. Theamino acid sequences deduced from SEQ ID NOs:25 and 26 were identical tothe amino acid sequence determined from the MF-3 protein. Therefore, itis clearly demonstrated that this cDNA fragment is an MF-3 gene.

13-b) Cloning of MF-3 cDNA

Plaque hybridization was carried out using the MF-3 cDNA fragment withabout 380 bp as shown by SEQ ID NO:25 obtained in Example 13-a) as aprobe according to the method described in Example 11-d). Six cloneswith strong signals out of positive clones were subjected to furtheranalysis. Specifically, E. Coli cells harbouring the plasmid which has aregion containing an MF-3 cDNA were obtained from these phages byautomatic subcloning in E. Coli. The plasmids were purified from theseE. Coli cells, and pMF3-1, which contained the longest fragment withabout 750 bp cDNA, was selected, and the base sequence of the cDNA wasthen determined. The base sequence is shown by SEQ ID NO:3 in SequenceListing, and the MF-3 gene encodes a polypeptide having an amino acidsequence as shown by SEQ ID NO:10 in Sequence Listing.

EXAMPLE 14 Cloning of Antigenic Protein MF-4 Gene from M. furfur

14-a) Amplification of MF-4 Gene by RT-PCR

The oligonucleotides MF4F1 and MF4F2 deduced from the N-terminal aminoacid sequence of the MF-4 protein described in Example 10 weresynthesized and purified to be used as primers for PCR. The basesequences of MF4F1 and MF4F2 are shown by SEQ ID NOs:27 and 28 inSequence Listing, respectively. An MF-4 cDNA fragment was amplified bycarrying out RT-PCR according to the method described in Example 11-b).MF4F1 and M13M4 primers were used in the first PCR reaction, and MF4F1and M13M4 primers were used in the second PCR reaction. As a result ofthe PCR reaction, a cDNA fragment with about 700 bp in length wasamplified. The base sequence of the cDNA fragment amplified is shown bySEQ ID NO:29 in Sequence Listing. The amino acid sequence deduced fromSEQ ID NO:29 was identical to the amino acid sequence determined fromthe MF-4 protein. Therefore, it is clearly demonstrated that this cDNAfragment is an MF-4 gene.

14-b) Cloning of MF-4 cDNA

Plaque hybridization was carried out using the MF-4 cDNA fragment withabout 700 bp as shown by SEQ ID NO:29 obtained in Example 14-a) as aprobe according to the method described in Example 11-d). Four cloneswith strong signals out of positive clones were subjected to furtheranalysis. Specifically, E. coli cells harbouring the plasmid which has aregion containing an MF-4 cDNA were obtained from these phages byautomatic subcloning in E. coli. The plasmids were purified from theseE. coli cells, and pMF4-4, which contained the longest fragment withabout 820 bp cDNA, was selected, and the base sequence of the cDNA wasthen determined. The base sequence is shown by SEQ ID NO:4 in SequenceListing, and the MF-4 gene encodes a polypeptide having an amino acidsequence as shown by SEQ ID NO:11 in Sequence Listing.

EXAMPLE 15 Cloning of Antigenic Protein MF-5 Gene from M. furfur

15-a) Amplification of MF-5 Gene by RT-PCR

DNAd on the N-terminal amino acid sequence of the MF-5 protein describedin Example 10, since the protein was thought to share homologies withDLDH, the oligonucleotide mixture MF5F1 encoding the amino acid sequenceGYVAAIKA DNAd on the above amino acid sequence and the DLDH amino acidsequence of other living organisms, and the oligonucleotide MF5R2corresponding to a highly homologous region (amino acid sequenceMLAHKAEE) when compared with DLDH amino acid sequences between otherliving organisms were synthesized and purified to be used as primers forPCR. The base sequences of MF5F1 and MF5F2 are shown by SEQ ID NOs:30and 31 in Sequence Listing, respectively. An MF-5 cDNA fragment wasamplified by carrying out RT-PCR according to the method described inExample 11-b). MF5F1 and M13M4 primers were used in the first PCRreaction, and MF5F1 and MF5R2 primers were used in the second PCRreaction. As a result of the PCR reaction, a cDNA fragment with about900 bp in length was amplified. The base sequence of the cDNA fragmentamplified is shown by SEQ ID NO:32 in Sequence Listing. The amino acidsequence deduced from SEQ ID NO:32 was identical to the amino acidsequence determined from the MF-5 protein. Therefore, it is clearlydemonstrated that this cDNA fragment is an MF-5 gene.

15-b) Cloning of MF-5 cDNA

Plaque hybridization was carried out using the MF-5 cDNA fragment withabout 900 bp as shown by SEQ ID NO:32 obtained in Example 15-a) as aprobe according to the method described in Example 11-d). Twelve cloneswith strong signals out of positive clones were subjected to furtheranalysis. Specifically, E. coli cells harbouring the plasmid which has aregion containing an MF-5 cDNA were obtained from these phages byautomatic subcloning in E. coli. The plasmids were purified from theseE. coli cells, and pMF5-6 and pMF5-7, which contained the longestfragment with about 1.6 kbp cDNA, was selected, and the base sequence ofthe cDNA was then determined. The base sequences are shown by SEQ IDNOs:5 and 33 in Sequence Listing, and the MF-5 gene encodes apolypeptide having an amino acid sequence as shown by SEQ ID NOs:12 and34 in Sequence Listing. These two kinds of genes have homology of 92% inthe base sequence, and 96% in the amino acid sequence encoding thereof,and were substantially identical to the amino acid sequence determinedfrom the MF-5 protein. Therefore, it is clearly demonstrated that bothof the genes are an MF-5 gene.

EXAMPLE 16 Cloning of Antigenic Protein MF-6 Gene from M. furfur

16-a) Amplification of MF-6 Gene by RT-PCR

The oligonucleotide mixtures MF6F1 and MF6F2 deduced from the N-terminalamino acid sequence of the MF-6 protein described in Example 10 weresynthesized and purified to be used as primers for PCR. The basesequences of MF6F1 and MF6F2 are shown by SEQ ID NOs:35 and 36 inSequence Listing, respectively. An MF-6 cDNA fragment was amplified bycarrying out RT-PCR according to the method described in Example 11-b).MF6F1 and M13M4 primers were used in the first PCR reaction, and MF6F2and M13M4 primers were used in the second PCR reaction. As a result ofthe PCR reaction, a cDNA fragment with about 1.0 kbp in length wasamplified. The amplified cDNA fragment was cloned into a pUC118 vector,and as a result, two kinds of cDNA having different cleavage patterns ofrestriction enzymes were detected. The base sequences of these cDNAfragments are shown by SEQ ID NOs:37 and 38 in Sequence Listing.Although these two genes have homology of 90% in the base sequence, and94% in the amino acid sequence deduced from the base sequence, they aredifferent genes. The amino acid sequences deduced from SEQ ID NOs:37 and38 were nearly identical to the amino acid sequence determined from theMF-6 protein described in Example 10. Therefore, it is clearlydemonstrated that this cDNA fragment is an MF-6 gene.

16-b) Cloning of MF-6 cDNA

Plaque hybridization was carried out using the MF-6 cDNA fragments withabout 1.0 kbp as shown by SEQ ID NOs:37 and 38 obtained in Example 16-a)as probes according to the method described in Example 11-d). Ten cloneswith strong signals out of positive clones were subjected to furtheranalysis. Specifically, E. coli cells harbouring the plasmid which has aregion containing an MF-6 cDNA were obtained from these phages byautomatic subcloning in E. coli. The plasmids were purified from theseE. coli cells, and pMF6-13, which contained the longest fragment withabout 1.2 kbp cDNA, was selected, and the base sequence of the cDNA wasthen determined. The base sequence is shown by SEQ ID NO:6 in SequenceListing, and the MF-6 gene encodes a polypeptide having an amino acidsequence as shown by SEQ ID NO:13 in Sequence Listing. Although thisgene lacks a encoding region of N-terminal amino acid sequence, it wasnearly identical to the cDNA fragment of MF-6 obtained in Example 16-a).Therefore, it is clearly demonstrated that this cDNA fragment is an MF-6gene.

EXAMPLE 17 Cloning of Antigenic Protein MF-7 Gene from M. furfur

17-a) Amplification of MF-7 Gene by RT-PCR

The oligonucleotide mixtures MF7F1 and MF7F2 deduced from the N-terminalamino acid sequence of the MF-7 protein described in Example 10 weresynthesized and purified to be used as primers for PCR. The basesequences of MF7F1 and MF7F2 are shown by SEQ ID NOs:39 and 40 inSequence Listing, respectively. An MF-7 cDNA fragment was amplified bycarrying out RT-PCR according to the method described in Example 11-b).MF7F1 and M13M4 primers were used in the first PCR reaction, and MF7F2and M13M4 primers were used in the second PCR reaction. As a result ofthe PCR reaction, a cDNA fragment with about 0.4 kbp in length wasamplified. The amplified cDNA fragment was cloned into a pUC118 vector.The base sequence of the cDNA fragment amplified is shown by SEQ IDNO:41 in Sequence Listing. The amino acid sequence deduced from SEQ IDNO:41 was nearly identical to the amino acid sequence determined fromthe MF-7 protein described in Example 10. Therefore, it is clearlydemonstrated that this cDNA fragment is an MF-7 gene.

17-b) Cloning of MF-7 cDNA

Plaque hybridization was carried out using the MF-7 cDNA fragment withabout 0.4 kbp as shown by SEQ ID NO:41 obtained in Example 17-a) as aprobe according to the method described in Example 11-d). Five cloneswith strong signals out of positive clones were subjected to furtheranalysis. Specifically, E. coli cells harbouring the plasmid which has aregion containing an MF-7 cDNA were obtained from these phages byautomatic subcloning in E. coli. The plasmids were purified from theseE. coli cells, and pMF7-1, which contained the longest with about 0.4kbp cDNA, was selected, and the base sequence of the cDNA was thendetermined. The base sequence is shown by SEQ ID NO:7 in SequenceListing, and the MF-7 gene encodes a polypeptide having an amino acidsequence as shown by SEQ ID NO:14 in Sequence Listing.

EXAMPLE 18 Synthesis of MF-1 Overlap Peptides and Deduction ofAntigen-Binding Sites

18-a) Synthesis of MF-1 Overlap Peptides

MF-1 overlap peptides were synthesized using a peptide synthesizer(PSSM-8, manufactured by Shimadzu Corporation). The entire amino acidsequence was covered by 33 kinds of peptides on the basis of thesequence of MF-1, as shown by SEQ ID NO:8 (FIG. 21), each peptideconsisting of 15 (16 or 17 in some cases) amino acid residues, and beingoverlapped with 10 amino acid residues.

First, a resin (50 mg) previously coupled with the Fmoc form of theC-terminal amino acid of each peptide (0.2 to 0.5 mmol/g resin) wastreated with 30% piperidine/DMF (0.5 ml) to remove the Fmoc group. Afterthe resin was washed with DMF (0.6 ml×5 times), the Fmoc form of thedesired amino acid activated with PyBOP and HOBt (used in DMF solutioncontaining the Fmoc in excess by 10 times relative to the amount of theC-terminal amino acid content) and an N-methylmorpholine/DMF solutionwere added, followed by a reaction at room temperature for 30 minutes.The resin was then washed with DMF (0.6 ml×5 times). This series ofprocedures were repeated in cycles until a peptide having the desiredsequence was obtained.

Next, this resin was admixed with a TFA-DNAd mixed solution (94% TFA, 5%anisole, 1% ethanedithiol (EDT)) (0.7 ml) and kept standing at roomtemperature for 2 hours (for tryptophan-containing peptides, a mixedsolution of TFA (94%), anisole (3%), EDT (3%), and 2-methylindole (5 mg)being used; for arginine-containing peptides, a mixed solution of TFA(82%), H₂O (5%), thioanisole (5%), EDT (3%), ethylmethyl sulfide (2%),and phenol (3%) being used; in the case for the arginine-containingpeptides, the resin was kept standing at room temperature for 8 hours).The resin was filtered off, and ethyl ether (14 ml) was added to thefiltrate to allow crystallization. The precipitated crystals wererecovered by centrifugation (3,000 rpm, 10 minutes) and washed withethyl ether, and they were then centrifuged again to remove thesupernatant, and the crystals were dried under reduced pressure. Theobtained crystals were assayed for its purity by reversed-phase HPLC. Inaddition, as occasion demands, the molecular weight was confirmed byLC-MS, and the crystals were purified by reversed-phase HPLC.

18-b) Identification of Binding Peptides to IgE Antibodies in Human Sera

Each of the peptides shown in FIG. 21 coated on a 96-well microplate at1 μg/well using a peptide coating kit (manufactured by Takara Shuzo Co.,Ltd.). A 2-fold dilution of each of 14 sera in total out of 13 sera frompatients with M. furfur RAST positive, and 1 pooled serum was added toeach well. After the reaction was carried out according to the manual, aβ-galactosidase-labeled anti-IgE antibody and then an enzyme substratewere added, followed by absorbance measurement at 415 nm. The absorbanceas used sera from normal individuals for 33 peptides was 20 on theaverage. A positive group was defined as those showing absorbance of notless than 40, which is 2-folds that of the sera from the normalindividuals. The positive group having absorbance of not less than 40was further classified into four ranks, and the results are shown inFIG. 22. The sera of patients with M. furfur RAST positive reactedstrongly to four to five kinds of peptide fragments.

18-c) Estimation of Epitopes of Mouse Monoclonal Antibodies Against MF-1

After three monoclonal antibodies against MF-1, i.e., M-40, MmAb37, andMAb51, were added to, and reacted with, microplates coated with each ofthe peptides of FIG. 21 described in Example 18-b), a peroxidase-labeledanti-IgG antibody and then an enzyme substrate were added, followed byabsorbance measurement at 450 nm. M-40 and MmAb37 reacted to Peptide 5,while MAb51 reacted to Peptides 25 and 26. In consideration of the abovefindings in combination with the results of FIG. 22, it was made clearthat these peptides contained B cell epitope.

EXAMPLE 19 Application of Recombinant Malassezia Antigenic Proteins forDiagnosis

19-a) Method for Measuring Specific IgE Antibodies by RAST Method

Activation of a paper disc with cyanogen bromide and coupling of therecombinant Malassezia antigenic protein to the paper disc were carriedout according to the method of Miyamoto et al. (Allergy, 22, 584-594,1973). One paper disc, previously coupled with the above antigenicprotein, and 50 μl of sera from patients were added to a polystyrenetube, followed by incubation at room temperature for 3 hours. The paperdisc was washed three times with a physiological saline containing 0.2%Tween 20, and 50 μl of the ¹²⁵I-labeled anti-human IgE antibody of theRAST-RIA kit, manufactured by Pharmacia, was added, followed byincubation at room temperature overnight. After the disc was washedthree times again, radioactivity was assayed using a gamma counter. TheIgE antibody titer was calculated from a standard curve drawn using areference reagent of the kit at the same time. Specimens yielding valuesexceeding the upper limit of the standard curve (>17.5 PRU/ml) werediluted 10 folds or 100 folds with equine serum and assayed again,followed by calculation of their antibody titer.

19-b) Diagnosis Using Recombinant Malassezia Antigenic Proteins rMF-1,rMF-2, and rMF-4

A skin test using the above antigenic proteins was performed on patientswith atopic dermatitis (hereinafter abbreviated AD) or bronchial asthma(hereinafter abbreviated BA) or both complications (AD+BA). Forty-threeout of 57 for the AD patients (75%), 108 out of 919 for the BA patients(12%), and 47 out of 102 for the AD+BA patients (46%) were positivepatients, showing a very high ratio for positive in the AD patients.Also, 100%, 59%, and 85% of these AD, BA, and AD+BA patients withpositive for skin tests, respectively, were positive in IgE antibodymeasurement by RAST method.

The IgE antibody titers for three kinds of the recombinant antigenicproteins rMF-1, rMF-2, and rMF-4 were assayed by RAST method (RIAmethod) on the 76 cases of patients with positive in the skin test usingthe above antigenic proteins and positive in RAST (1 or higher score)(AD: 30 patients, BA: 20 patients, AD+BA: 26 patients) as an object formeasurement. The IgE antibody titers for the above antigenic proteinswere assayed in the same manner on 12 negative individuals in the skintests (normal individuals). As a result, it was made obvious that theIgE antibodies against antigenic proteins were present in the sera frompatients at very high ratios. Especially, it was found that ratios ofpositive for rMF-1 and rMF-2 were high. In addition, surprisingly, theIgE antibody titers were very high. And especially in the case of the ADpatients, the IgE antibody titers were 100 PRU on average, with valuesexceeding 1,000 PRU in some patients. Also, the IgE antibody against anyone of the recombinant antigenic proteins rMF-1, rMF-2, and rMF-4 waspresent in the sera from all patients with RAST-positive for theMalassezia antigens.

INDUSTRIAL APPLICABILITY

According to the present invention, there can be provided an isolatedand purified antigenic protein having high purity from Malassezia,antigenic fragments thereof, and a specific antibody against thoseantigenic protein or fragments thereof. In addition, there can beprovided a diagnostic agent, a therapeutic agent, or a prophylactic drugfor Malassezia allergoses, wherein the agent includes, as an activeingredient, the antigenic protein or fragments thereof.

Further, according to the present invention, there can be provided anovel recombinant Malassezia antigenic protein, genes encoding theantigenic protein, and an epitope of the antigenic protein.

1. An isolated and purified antigenic protein from fungi of the genusMalassezia, wherein said antigenic protein has a partial amino acidsequence as shown by SEQ ID NO: 45, and has a molecular weight of about21 kDa as determined by SDS-PAGE under reduced conditions and about 40kDa as determined by SDS-PAGE under non-reduced conditions, anisoelectric point of about 4.7 in a native state, and an isoelectricpoint of about 5.3 in a denatured state with 8 M urea.
 2. The antigenicprotein according to claim 1, wherein said antigenic protein has theamino acid sequence as shown by SEQ ID NO:
 8. 3. A diagnostic agent forMalassezia allergies or Malassezia infectious disease, wherein saiddiagnostic agent includes, as a biologically active ingredient, theantigenic protein according to claims 1 or
 2. 4. A therapeutic agent forMalassezia allergies or Malassezia infectious disease, wherein saidtherapeutic agent includes, as a biologically active ingredient, theantigenic protein according to claims 1 or 2.